Difference between revisions of "Team:USTC/Notebook"

Revision as of 07:26, 13 September 2015

Protocols
Daily Notes-Stage 1
Daily Notes-Stage 2
Raw Data

Protocols

Protocols

2015 4/7

Medium preparation

Prepare and keep a part of solid medium and broth in the refrigerator as a backup.

Cell culture

Culture the cell,TOP10, with LB liquid solution overnight.

2015 5/7

Design protocol

Take the solid medium, heat it until liquid. Then make two plate of solid medium, evenly. Cool it to the room temperature.
prepare antibiotic solution with concentration gradient
a. prepare antibiotic(neomycin) solution with ddH2O at 50mg/ml as mother solution 1. And dilute it to 10% concertration(5mg/ml) with ddH2O as mother solution 2. Then prepare 8 parts of solution as this:

Number	Solution 1(V/uL)	Solution 2(V/uL)
0	0	0
1	0	0.2
2	0	1
3	0.2	0
4	0.4	0
5	0.6	0
6	0.8	0
7	1	0

Keep them in the 70℃ water bath. Then add 800uL liquid solid medium into each Ep tubes. Transfer them into 50℃ water bath.

Cut 4 hole in each plate of solid medium, whose diameter is at 8mm.
Take 5mL culture into the 2 Ep tubes. Centrifuge it at twice. Then add liquid solid medium at 50℃, each 800uL, dissolve evenly.
Take 200uL top10 solution into 8 Ep tubes each at 50℃. Then add the mixed solution into the hole at once until the hole is added up.

Results: the raw results showed differerent characteristics of chemotaxis based on distinct antibiotic concentration.
0-3
4-7

Analysis: 其中3号有最明显的趋化结果，说明这个趋化结果并不是完全正相关或负相关的。但是结论可能是存疑的，过量的加入固体培养基会导致可能的菌液溢出从而形成如此的趋化形状。因此实验配方需要进行更改。

2015 6/7

今天在延续着昨天的实验思路，利用灭菌的1mL枪头打洞，然后将含抗培养基和菌液混合，打入洞中，待凝固后放入恒温箱培养。
实验结果:这次并没有长出相应的环带。有可能是由于在放入之前被紫外灯照射了的缘故导致的细菌死亡。但是同时也有可能是由于孔洞较深，使得大肠杆菌没有办法从边缘逃逸。
2015 7/7

前一天的培养基里头出现了E.coli,但是伴随着杂菌。
于是重复了7月6日的实验，此次打洞是，为了方便控制液体体积，这会使用的是灭菌的200uL枪头打洞。不过由于体积过小，导致仍有一部分液体溢出。
2015 8/7

发现7月7日的结果与7月5日结果有差别，并且各孔的趋化效果呈振荡形式，并且环的效果不是很明显。

analyse：形成环的一部分孔洞是存在着溢出情况的，某种程度上印证的之前关于溢出的猜测。
可能E.coli的运动能力不是很强，引起环的明显程度不够，或者是由于抗生素的浓度过高，而杀死或抑制了细菌。后面实验还需要重新审核实验方案，并且需要克服由于加样无法精确掌握体积的问题。

接下来的思路：可以考虑利用含有抗性的菌来做趋化实验，但是首先要求该种抗性仅仅是抗生素作用靶点的改变，而不是分解或者是抗生素失活的方式，以免改变抗生素的局部浓度。
2015 9/7
转化

今天转化的是来自刘玉婷实验室的TG1感受态细胞，感受态细胞是由刘玉婷与张启钧提前完成的，主要目的是为了检测：
- 感受态细胞是否高效
- 感受态细胞是否污染
实验流程：
1. 冰上放置感受态细胞2~3min，一共4管
2. 加入5ul检测质粒，其中3管有100ul感受态细胞两支，200ul感受态细胞两支，其中100ul的感受态一支加入5ul质粒，一支加入1ul质粒，在冰上放置30min
3. 在42℃水浴锅中热激45s
4. 冰上放置3min
5. 加入900ulLB培养基培养55min
6. 离心12000g 15s，放弃上清液，加入200ulLB培养基
7. 涂板，板带有氨苄抗性
8. 培养20h观察实验结果
绿脓杆菌接菌

从金范老师那里拿来的绿脓杆菌生长完整，选取了其中的三个菌落挑单，发现绿脓杆菌单菌落相比大肠杆菌较大，培养基有异味。取5mL LB培养基直接将挑好的细菌用枪头打在其中培养16h次日以备使用。
2015 10/7
冻存绿脓杆菌

绿脓杆菌需要冻存，冻存的最后甘油体积分数为15%

灭菌时配好的50%甘油进行灭菌，因为根据小木虫的经验帖，100%体积的甘油不宜直接灭菌

甘油的密度为：1.26g/mL

50%甘油（50mL）配方为：
- 25mL 蒸馏水
- 25mL（31.5g）甘油
混匀之后即可灭菌，冻存管同时灭菌

15%冻存甘油管的配方为（1mL）：
- 700ul 培养菌液
- 300ul 15%甘油溶液
冻存于-40℃冰箱中，备用

涂板绿脓杆菌

早上：抽取培养过的绿脓杆菌取100ul加入LB固体培养基中进行涂板

结果发现，细菌密度过浓，没有单菌落形成

晚上：抽取培养的绿脓杆菌，进行1：4的培养基稀释，之后取100ul加入到LB固体培养基中涂板，过夜培养

转化

转化材料是来自洪炯老师的XL21 Gold感受态细胞，和昨日刘玉婷制备的TG1感受态细胞，希望进一步证明感受态细胞没有被污染，以及扩增pSBC1C3质粒，和T7启动子，tRNA part

实验流程：
1. 冰上放置感受态细胞3min，一共4管
2. 稀释parts（T7启动子：2013年Kit5的6N孔，tRNA：2015年Kit5的21I孔），加入10ul灭菌过的蒸馏水，稀释混匀。
3. 加入1ul检测T7启动子与tRNA的parts至XL21 gold感受态细胞，加入5ulpSB1C3至XL21 Gold感受态细胞，在冰上放置30min
4. 在42℃水浴锅中热激55s
5. 冰上放置3min
6. 加入到5mL的氯霉素抗性的液体培养基。
7. 培养12h明日观察结果（21：00完成）
1st Colonial PCR for COMPX
- 2μl Taq buffer with KCl
- 0.4μl dNTPs
- 1μl COMPX-F Primer
- 1μl COMPX-R Primer
- 0.25μl Taq DNA Polymerase
- 2μl MgCl2
- 1μl TOP10 Solution
- 12.35μl ddH2O
Renaturation at 45℃, extend for 1 min.

1st Electrophoresis for Colonical PCR

No band except marker resulted due to Gelred past due.
2015 11/7
重新转化实验

氯霉素的浓度昨天有误，今天重新进行配置，再养菌

重新制备感受态细胞

感受态细胞用Amp进行空白测试

重新对绿脓杆菌涂板

涂板稀释浓度增加到1:10与1:15，因为绿脓杆菌浓度过高，单菌落太少，不宜储存

2nd Colonial PCR for COMPX
- 2μl Taq buffer with KCl
- 0.4μl dNTPs
- 1μl COMPX-F Primer
- 1μl COMPX-R Primer
- 0.25μl Taq DNA Polymerase
- 2μl MgCl2
- 1μl TOP10 Solution
- 12.35μl ddH2O
Renaturation temperature varies from 50℃ to 60℃, extend for 1 min.

2nd Electrophoresis for Colonical PCR

Add Gelred in loading buffer. The band of PCR product appears in all samples.

结果如下图所示：

意外出现问题，由于胶用水去溶解了，导致Marker都没有跑开

该结果则较好的显示出了菌落PCR成功的p出了COMPX蛋白

教训：
- 误用琼脂
- 误用水区溶解胶
- 电压需要调整回90V，30min
3rd Colonial PCR for COMPX
- 2μl Taq buffer with KCl
- 0.4μl dNTPs
- 1μl COMPX-F Primer
- 1μl COMPX-R Primer
- 0.25μl Taq DNA Polymerase
- 2μl MgCl2
- 1μl TOP10 Solution
- 12.35μl ddH2O
Renaturation at 55℃, extend for 1 min.

3rd Electrophoresis for Colonical PCR

No results again.

1st Transformation

1 Add 1μl BBa K1492002 or 5μl pSB1C3 plasmid to competent cells.

2 Heat at 42℃ for 45 s by mistake.

3 Incubate on ice for 30 min.

4 Heat at 42℃ for 45 s.

5 Incubate on ice for 3 min.

6 Add 900 μl LB medium, incubate at 37℃ for 1 h.

7 Centrifuge and discard 85 μl LB medium.

8 Resuspend cells and add 100 μl to LB medium with chloramphenicol. Incubate at 37℃.
2015 12/7
4th Colonial PCR for COMPX
- 2μl Taq buffer with KCl
- 0.4μl dNTPs
- 1μl COMPX-F Primer
- 1μl COMPX-R Primer
- 0.25μl Taq DNA Polymerase
- 2μl MgCl2
- 1μl TOP10 Solution
- 12.35μl ddH2O
Renaturation at 55℃, extend for 1 min. 2 tubes without Taq DNA Polymerase as blank control.

4th Electrophoresis for Colonical PCR

Bands are predicted except one. Maybe caused by accident. PCR product keeped for later use.

1st Plasmid Extraction for BBa K1492002 & pSB1C3

1 Centifuge 5 ml bacterium solution, few sediment getted.

2 Add 250 μl Buffer P1, resuspend cells.

3 Add 250 μl Buffer P2, mix well, 4 min's standing.

4 Add 350 μl Buffer P3, mix well.

5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.

6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.

7 11.6 rpm centifuge 1 min.

8 Add 50 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.

1st Electrophoresis for Plasmid Extraction

No result. May due to high concentration of chloramphenicol, bacteria grow very slow. As there is few bacteria, no plasmid getted. Bacterium solution dilute 1:1 with LB medium.

绿脓杆菌划线

大约在傍晚7点左右，利用划线法在LB平板上培养绿脓杆菌。一块是从昨天的培养的培养基里分出来的，另一块是从菌液中沾取划线的。

10点离开前，看了一次，从培养基分出来的那一块平板已经有菌落生成了。
2015 13/7
The steak-plate of Pseudomonas aeruginosa

By using the steak-plate method, we successful separated the single conlony of P. aeruginosa.
\(^o^)/~！

2nd Plasmid Extraction for BBa K1492002 & pSB1C3

1 Centifuge 4.5 ml bacterium solution.

2 Add 250 μl Buffer P1, resuspend cells.

3 Add 250 μl Buffer P2, mix well, 4 min's standing.

4 Add 350 μl Buffer P3, mix well.

5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.

6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.

7 11.6 rpm centifuge 1 min.

8 Add 30 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.

2nd Electrophoresis for Plasmid Extraction

Successfully extracted tRNA plasmid. Failed to extract pSB1C3 plasmid, cause culture is contaminated.
Result:

tRNA 114ng/μl

1st PCR for tRNA
- 2μl Taq buffer with KCl
- 0.4μl dNTPs
- 1μl tRNA-F2 Primer
- 1μl tRNA-R Primer
- 0.25μl Taq DNA Polymerase
- 2μl MgCl2
- 1μl tRNA plasmid
- 12.35μl ddH2O
Renaturation at 50℃, extend for 1:30.

1st Electrophoresis for tRNA PCR

No result.

Transformation of Four Parts of Plate 4
1. material:
  competent cell TG1 from the lab 319
  4 parts of the plate 4(pSB1A2)
  No.1:BBa_B0034 No.2:BBa_C0051
  No.3:BBa_R0051 No.4:BBa_E0040
2. the procedure
  (1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
  (2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
  (3)Put the tubes on the ice about 30 mins.
  (4)Make a heat shock at 42 degree centigrade about 60 sec
  (5)Put the tubes on the ice about 3 mins again.
  (6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
  (7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
  (8)Discard the supernatant liquid and leave about 100ul medium.
  (9)Coat plate: add 100ul solution in a plate
2015 14/7
The Results of Transformation of Jul 13th

transformations of three parts are successful:
No.1 B0034
No.2 C0051
No.4 E0040
transformation of one part is failing:
No.3 R0051

Plasmid Extraction of pSB1C3

the plasmis:pSB1C3

the procedure:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg.
PS:this protocol is from lab 319

the concentration :11.0ng/ul
PS:the concentration of the control plasmid is 136.0 ng/ul,so we guess that maybe the concentration of bacterium solution is low.Also we guess that it's caused by the missing of spreading.

Transformation of Three Parts and One Plasmid
1. the plasmid going to be transformed:
  No.1 BBa_R0062(plate 2;pSB1C3)
  No.2 BBa_B0015(plate 3;pSB1C3)
  No.3 BBa_R0051(plate 4;pSB1A2)
  No.4 pSB1C3
2. the procedure:
  note:add 1 ul R0062 ,1 ul B0015,2 ul R0051,
  2 ul pSB1C3 to 100 ul competent cells respectively
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate

Plasmid Extraction of Three Parts of Plate 4

the procedure is the same as before
the concentration :
B0034 157.6ng/ul, 113.7ng/ul;
C0051 171.7ng/ul, 194.6ng/ul;
E0040 278.5ng/ul, 228.2ng/ul
2015 15/7
Results of transformation

R0051 no colony
R0062 have single colony
B0015 have single colony
pSB1C3 have single colony and the colony is red

Plasmid Extraction Results

This is extraction was for previous transformation.
- pSB1C3(1): 108ng/uL
- pSB1C3(2): 157ng/uL
- B0015(1): 19ng/uL
- B0015(2): 93ng/uL
Prepared for further use.

2015 16/7

Colonical PCR for micF and SoxS

Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
micF-luxI-R	TTATAGTCATATAAATTTTCTCCTCTTTCCGAATGCGAGGCATCC
micF-F	GCGGCCGCTTCTAGAGCCTCATTAATCAGTCGGC
SoxS-RBS-luxI-R	TATAGTCATATAAATTTTCTCCTCTTTCTAGTGCGTGCGCCGGTACCTT
BB-SoxS-F	CGGCCGCTTCTAGAGCGTGCGGAACATTCG

For micF, primers including micF-luxI-R, micF-F, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl micF-luxI-R Primer
1μl micF-F Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Top10(K-12) Strain E. coli bacterial solution
12.35μl ddH2O

For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl SoxS-RBS-luxI-R Primer
1μl BB-SoxS-F Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl 1μl Top10(K-12) Strain E. coli bacterial solution
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Characterization of Colonical PCR by Gel Electrophoresis

PCR
The result illustrated the success of PCR

2015 17/7

Colonical PCR for OprF from Psudomonus aeruginosa

Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
OprF-F	AGAGGAGAAAATCGGAATGAAACTGAAGAACAC
OprF+Ter-R	TGATGCCTGGCTCTAGTATTACTTGGCTTCAGCTT

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl OprF-F Primer
1μl OprF+Ter-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Psudomonus aeruginosa (PAO1) bacterial solution
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Plasmid Extraction of SCVE

SCVE synthesized from Sangon Biotech Co.,Ltd.

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg.

Transformation for CRISPR

1.材料：competent cell TG1 from the lab 319，4Mparts of the plate 4(pSB1A2)

2.编号：1:BBa_B0034 2:BBa_C0051

3:BBa_R0051 4:BBa_E0040

3.步骤：

（1）将感受态细胞置于冰上，孵化5分钟；

（2）取1ul 含代转parts的plasmid与感受态细胞混合，轻轻混匀；

（3）冰上放置30min，中间轻摇数次，防止菌体沉淀；

（4）42.0度水浴静置热激90s（这个时间控制很重要），立即放入冰上冷却2-3min；

（5）加入900ul LB(不含抗生素)培养基，混匀；

（6）37度振荡培养1h；

（7）12，000rpm 离心15s，用移液器小心吸出850ul上清丢弃后，保留50ul重悬菌液;

（8）用移液器混匀菌液，均匀涂布于含抗生素（Cl 200ml LB:50ul 100ng/ul Cl）的平板上，放入37度培养箱倒置培养过夜。

Characterization of SCVE and OprF by Gel Electrophoresis

PCR
Only 1/4 PCR results successfully duplicated results, plasimid extraction from SCVE showed well.

Second PCR for OprF didn't work well:(

Digestion of E0040(EGFP)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

Nuclease-free water: 15ul
E0040: 1ul
10* FastDigest Green Buffer: 2ul
EcoRI: 1ul
SpeI: 1ul

We are trying to figure out the best conditions of our experiment and whether we need loading buffer

Time	With Loading Buffer or not
5min	Yes(1), No(2)
2h	Yes(3), No(4)

PCR of E0040(EGFP)

Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
E0040-F	ATGCGTAAAGGAGAAGAACTTT
R0051-E0040-R	TACGCATATAAATTTTCTCCTCTTTGCAACCA

For E0040, primers including E0040-F, R0051-E0040-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl E0040-F Primer
1μl R0051-E0040-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl plasmid(pSB1C3) containing E0040
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Attention: R0051-E0040-R Primer is not appropriate because of only 8 complementary base pairs.

Characterization of E0040 from Digestion and PCR

PCR
Result illustrated that We do need loading buffer, and 5 min do work!

PCR showed more concentration of E0040!

Gel Extraction

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Final OD results:

Code	OD(ng/ul)
PCR(1)	15.3
PCR(2)	4.5
PCR(3)	27.0
Digestion(1) with 5 mins	15.4
Digestion(3) with 2 h	22.7

Perseved for further use.

2015 18/7

Transfofmation of Three Parts

Protocol as following

100μl (three tubes)competent cells
C0062(pSB1C3)
R0010(pSB1C3)
C0061(pSB1C3)
LB without antibiotic
LB medium with Amp/CmR

Plasmid Extraction of E0040

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Results

Code	OD(ng/ul)
E0040(1)	166.7
E0040(2)	169.2

Colonical PCR for OprF from Psudomonus aeruginosa

PCR protocol:
For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl OprF-F Primer
1μl OprF+Ter-R Primer
0.5μl Taq DNA Polymerase(add more 0.25ul then previous protocol)
2μl MgCl2
1μl Psudomonus aeruginosa (PAO1) bacterial solution
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Digestion of E0040(EGFP)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

Nuclease-free water: 15ul
E0040: 1ul
10* FastDigest Green Buffer: 2ul
EcoRI: 1ul
SpeI: 1ul

Characterization of OprF and E0040

图片名称
酶连效果不是很好，基本建议是改善昨日胶回收的条带进行PCR扩增，之前引物设计担心不是很好，但是看样子没有其他位点被p了出来；
OprF的PCR依然是在相同条件下只有一个条带p了出来，比较异常；此外引物二聚体较多，因此对此批进行了胶回收，以备后续使用。

Plasmid Extraction of Cas9(K1218011)

The bacteria were cultured at 9 am from successfully transformed bacteria.

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Characterization of Cas9

图片名称
转化成功，明天写实验结果讨论

2015 19/7

Digestion of K1218011(Cas9)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

Nuclease-free water: 15ul
K1218011: 1ul
10* FastDigest Green Buffer: 2ul
EcoRI: 1ul
SpeI: 1ul

Incubate into 37 degree celcius, 2h

Digestion of SCVE

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

Nuclease-free water: 16ul
SCVE: 1ul
10* FastDigest Green Buffer: 2ul
SmaI: 1ul

Incubate into 37 degree celcius, 2h

Colonical PCR for OprF from *Psudomonus aeruginosa, micF and SoxS from K-12

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl OprF-F Primer
1μl OprF+Ter-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Psudomonus aeruginosa (PAO1) bacterial solution
12.35μl ddH2O

For micF, primers including micF-luxI-R, micF-F, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl micF-luxI-R Primer
1μl micF-F Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Top10(K-12) Strain E. coli bacterial solution
12.35μl ddH2O

For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl SoxS-RBS-luxI-R Primer
1μl BB-SoxS-F Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl 1μl Top10(K-12) Strain E. coli bacterial solution
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Praparation for Competent Cell

protocol as the following:

Pick out two single colonies(TG1) and cultivate bacteria in 5mL LB media at 37 degree centigrade about 12 hours with shocking.
Take 5mL nutrient solution into 100 mL media and cultivate bacteria at 37 degree centigrade about one hour and a half with shocking till the OD660 reaches 0.4-0.5.
Absorb 50 mL nutrient solution into EP tubes with ice-bath about 10 mins.
Centrifuge the tubes at 4100xg at 4 degree centigrade about 10mins and discard the supernatant liquid.
Utilize 30 mL 0.1M calcium chloride to suspend bacteria.
Centrifuge the tubes at 4100xg at 4 degree centigrade about 10 mins and discard the supernatant liquid.
Utilize 2 mL 0.1M calcium chloride with 15% glycerol to suspend bacteria and then ready for transformation quickly or restore at -70 degree centigrade.

Time(min)	OD600
40	0.195
60	0.203
80	0.309
90	0.439

PS:dilute the solution by adding 100 mL LB media after first test.

2015 20/7
Negative Chemotaxis Characterization

term 1, material:
- LB solid medium plates x4
- 1g/ml Chloramphenicol
- E.coli Top10
- filter paper
Plasmid Extraction of EmrE and ArcB

done by Wang Luyao

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

the concentration as following
EmrE 70.2 ng/ul 92.3 ng/ul
ArcB 62.6 ng/ul 95.2 ng/ul

Transformation of R0051 and C0061

the source of parts:
No.1 R0051 2014 kit plate 4
No.2 R0051 2014 kit plate 4
No.3 control
No.4 C0061 2015 kit plate 4
No.5 C0061 2015 kit plate 4
No.6 C0061 2014 kit plate 4

No.1 and No.4 the competent cell is from lab319
No.2 and No.5 the competent cell is made by us (firt)
No.3 and No.6 the competent cell is made by us (second)

the protocol as following
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate

PCR of OrpF

material:
- 13 uL Taq Master Mix
- 11 uL dd water
- 1 uL PAO1 bacterial
- 0.5 uL primer Fd(OrpF-F)
- 0.5 uL primer Rv(OrpF+Ter-R)
  PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.
Renaturation at 50℃, extend for 1:10
2015 21/7
PCR of OprF

material:
No.1 and No.2:
- 6.5 uL Taq Master Mix
- 1 uL PAO1 bacterial solution
- 4.5 uL dd water
- 0.5 uL OprF-F
- 0.5 uL OprF+Ter-R
No.3 and No.4:
- 6.5 uL Taq Master Mix
- 4 uL OprF PCR product
- 1.5 uL dd water
- 0.5 uL OprF-F
- 0.5 uL OprF+Ter-R
PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.

Renaturation at 55℃, extend for 1:10

OprF for PCR and gel electrophoresisOprF

analyze: the length of the product is

2015 22/7

Preparation of Semi-Solid LB Medium

In order to characterization the movement of TOP10, semi-solid LB medium preparation is indispensable in this research.

(500mL Protocol)

Ingredients	Mass	Proportion(g/mL)
Yeast Extract	2.5g	0.5%
Trtptone	5g	1%
NaCl	5g	1%
Agar	1.25g	0.25%

Compared with solid LB medium, containing 1.5% agar in there.

Plasmid Extraction C0051,B0015 and R0062

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

the concentration as following
C0015:171.7 ng/uL
B0015:93 ng/uL
R0062:144.3 ng/uL

PCR of C0051,B0015 and R0062

material:
C0051 plasmid(171.7 ng/uL)
B0015 plasmid(93 ng/uL)
R0062 plasmid(144.3 ng/uL)
all the solutions are diluted 10-folds

Primers produced from Sangon Biotech Co.,Ltd.

Primers	Sequence(5' to 3')
C0051-F(I)	att tat atg agc aaa aag aaa cca tta tca
C0051-B0015-R(I)	tat ttg atg cct ggc tct agt gat cta cac tag cac ta
B0015-R(I)	gcc gct act agt taa acg cag aaa ggc cca cc
B0015-F	cca ggc atc aaa taa aac gaa agg
R0062-F	gcg gcc gct tct aga gac ctg tag gat cg
R0062-C0051-R	tgt gct cat ata aat ttt ctc ctc ttt cta gat ttt att cga c

No.1 and No.2 for C0051(803bp)
Protocol as this:

2.5 μl Taq buffer with KCl
2.5 μl dNTPs
0.75 μl C0051-F Primer
0.75 μl C0051-B0015-R(I) Primer
0.5 μl Taq DNA Polymerase
1.0 μl MgCl2
2 uL C0051 solution
15 μl ddH2O

No.3 and No.4 for B0015(142bp)
Protocol as this:

2.5 μl Taq buffer with KCl
2.5 μl dNTPs
0.75 μl B0015-F Primer
0.75 μl B0015-R(I) Primer
0.5 μl Taq DNA Polymerase
1.0 μl MgCl2
3 uL C0051 solution
14 μl ddH2O

No.5 and No.6 for R0062(104bp)
Protocol as this:

2.5 μl Taq buffer with KCl
2.5 μl dNTPs
0.75 μl R0062-F Primer
0.75 μl R0062-C0051-R Primer
0.5 μl Taq DNA Polymerase
1.0 μl MgCl2
2 uL R0062 solution
15 μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)
4 celcius degree for preserveation.

Preparation Semi-Solid M63 Medium

In order to characterization the movement of TOP10, semi-solid M63 medium preparation is indispensable in this research.

(500mL Protocol)

Ingredients	Mass	Proportion(g/mL) or Mole
Yeast Extract	2.5g	0.5%
Trtptone	5g	1%
NaCl	5g	1%
Agar	1.25g	0.25%
KH2PO4	6.8g	50mM
KOH	2.1g	37.5mM
(NH4)2SO4	0.09g	7.5mM
MgSO4	0.06g	0.5mM
FeSO4·7H2O	0.2710g	1.95mM
D-Glucose	1.98g	11mM

Colonical PCR for OprF from Psudomonus aeruginosa and SCVE from pUC57

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl OprF-F Primer
1μl OprF+Ter-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Psudomonus aeruginosa (PAO1) bacterial solution
12.35μl ddH2O

For SCVE, Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
SCVE-T-R	TTGATGCCTGGCTCTAGTACACCAGCAGATCCGG
T7-RBS-SCVE-F	TTTTTGCATACTAGAGAAAGAGGAGAAAATTTATATGTATAGCTTTGTG

Protocol as this:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl SCVE-T-R Primer
1μl T7-RBS-SCVE-F Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl SCVE solution from plasmid extraction
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Gel Electrophoresis of OprF, SCVE micF and SoxS

micF and SoxS
图片名称

OprF and SCVE
图片名称

Gel Extraction

Material including: OprF, SoxS, micF

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

2015 23/7

Plasmid Extraction of R0062

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

PCR for OprF and C0061

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl OprF-F Primer
1μl OprF+Ter-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl Psudomonus aeruginosa (PAO1) bacterial solution
12.35μl ddH2O

For SCVE, Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
C0061-F	ATGACTATAATGATAAAAAAATCGGATTTTTTGGCA
C0061-B0015-R	ATTTGATGCCTGGGAGATCTACACTAGC

Protocol as this:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl C0061-F Primer
1μl C0061-B0015-R Primer
0.25μl Taq DNA Polymerase
2μl MgCl2
1μl C0061 solution
12.35μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Gel Extraction of C0062,B0015 and R0062

Material including: C0051,B0015 and R0062

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

the concentration as following:
C0051:12.7 ng/uL
B0015:7.3 ng/uL
R0062:7.7 ng/uL

PCR of OprF

material:

13 uL Taq Master Mix
11 uL dd water
1 uL PAO1 bacterial
0.5 uL primer Fd(OrpF-F)
0.5 uL primer Rv(OrpF+Ter-R)

Renaturation at 55℃, extend for 2min

Gel Electrophoresis Characterization

C0051 and B0015
图片名称

OprF
图片名称
That was relatively successully extracted OprF from colonical PCR.

R0062
图片名称

2015 24/7

Preparation for Homologous Recombination: Plasimid Digestion

Enzyme from Thermo Scientific FastDigest Enzyme with 10X Green Buffer

Protocol as following:

Nuclease-free water: 15ul
pSB1C3: 1ul
10* FastDigest Green Buffer: 2ul
EcoRI: 1ul
SpeI: 1ul

Incubate solution at 37 degree celcius about 2h.

PCR of C0061,B0015 and micF

material:
C0061 plasmid: 477.1 ng/uL(diluted 20-folds)
B0015 PCR product:7.3 ng/uL
micF-1 PCR product:13.4 ng/uL

ForB0015 Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
B0015-F	cca ggc atc aaa taa aac gaa agg
B0015-R(I)	gcc gct act agt ata taa acg cag aaa ggc cca cc

protocol:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl Primer-Fd
1μl Primer-Rv
0.25μl Taq DNA Polymerase
2μl MgCl2
1uL C0061 plasmid,4uL B0015 plasmid,2uL plasmid
(13.35-X)μl ddH2O

Renaturation at 55℃, extend for 1min

2015 25/7

PCR of C0061,B0015,R0062,R0010 and C0062

material:
C0061 plasmid:477.1 ng/uL(0.2 uL)
B0015 plasmid:19 ng/uL(3 uL)
R0062 plasmid:143.3 ng/uL(0.5 uL)
C0051 plasmid:171.7 ng/uL(0.5 uL)
R0010 plasmid:357.7 ng/uL(diluted 10-folds,1 uL)
C0062 plasmid:739.4 ng/uL(diluted 10-folds,1uL)
for R0062.R0010 and C0062Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
R0062-F	gcg gcc gct tct aga ctg tag gat cg
R0062-C0051-R	tgt gct cat ata aat ttt ctc ttt cta gat ttt att cga c
backbone-R0010-F	ccgctt cta gag cca tac gca aac cgc ctc tc
C0062-B0034-R	cta gtg tc agt aat aaa ttt tct cct ctt tg tgt gaa att gt
B0034-C0062-F	tat tac tga gca cta gat gaa aaa cat aaa tgc cga c
B0015-C0062-R	atg cct vggc tct agt gat cta cac tag cac t

protocol:

2μl Taq buffer with KCl
0.4μl dNTPs
1μl Primer-Fd
1μl Primer-Rv
0.25μl Taq DNA Polymerase
2μl MgCl2
XuL plasmid
(13.35-X)μl ddH2O

Renaturation at 55℃, extend for 1min
PS:
C0061:656bp B0015:142bp R0062:104bp
C0051:803bp C0062:813bp R0010:243bp

Characterization of micF and SoxS by Gel Electrophoresis

图片名称
micF were successfully PCRed.

Characterization of pSB1C3 and SoxS by Gel Electrophoresis

图片名称

2015 26/7
Characterization of R0051, C0061 and C0051 by Gel Electrophoresis

Plasimid Extraction for pSB1C3 and C0061

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Gel Extraction

Material including: R0051, C0051 and C0061

Preparation:
1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.
Procedure:
1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.
Characterization of B0015 and R0051 by Gel Electrophoresis

2015 27/7

Overlap Circuit

图片名称
This sensing circuit including promoter micF and SoxS.

Overlapping Fragments

In order to ligate the fragements micF or SoxS, C0061 and B0015, we need to make the equal mole proportion.

The mole of DNA molecule is calculated in the following ways:

图片名称

among which, c is the concentration of DNA, V is the volumn required and n is the length of DNA. Because of the same molar mass of As, Gs, Cs and Ts, computation of same molar mass is omitted.

According to our sample extracted from PCR and calculation, ingradients characterize as below:

Name	Concentration(ng/uL)	Length(bp)
micF	12.6	358
SoxS	27.5	705
C0061(LuxI)	18.5	656
B0015(Terminator)	9.5	143
Total		1161

The theoratical volumn proportion is:

SoxS-C0061-B0015: 5: 7: 3
micF-C0061-B0015: 5.5: 7: 3

Firstly, to ligate these parts, we need 10 cycles without primer:
for circuit SoxS-C0061-B0015:

Name	Volumn(uL)
SoxS	5
C0061	7
B0015	3
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
10X buffer with KCl	2
ddH2O	0.35

for circuit micF-C0061-B0015:

Name	Volumn(uL)
micF	5.5
C0061	7
B0015	3
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
10X buffer with KCl	2

PCR cycle including:

Preheat: 94 degree celcius, 5min
10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Then, we extracted 5 ul as template, and 15 ul adding with primer:
Primers were synthesized from Sangon Biotech. Co.,Ltd.

Primers	Sequence(5' to 3')
micF-F	GCGGCCGCTTCTAGAGCCTCATTAATCAGTCGGC
B0015-R	GCCGCTACTAGTATATAAACGCAG
BB-SoxS-F	CGGCCGCTTCTAGAGCGTGCGGAACATTCG

5ul-template protocol:

Name	Volumn(uL)
Template	5
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
micF-F(BB-SoxS-F)	1
B0015-R	1
10X buffer with KCl	2
ddH2O	8.35

15ul-protocol:

Name	Volumn(uL)
Solution	15
micF-F(BB-SoxS-F)	1
B0015-R	1
10X buffer with KCl	2
ddH2O	1

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

PCR of Five Parts

material:
R0010 plasmid:193.1ng/uL
C0062 plasmid:952.5ng/uL
B0015 plasmid:93ng/uL
R0062 plasmid：143.3ng/uL

For R0051 Primers produced from Sangon Biotech Co.,Ltd.

protocol:

2uL 10X buffer with KCl
0.4uL dNTP
1uL primers-F
1uL primers-R
0.25uL Taq Polymerase
2uL MgCl2
1uL plasmid
12.35 uL ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Overlap PCR of bacteria III

material:

R0062 PCR product:21.2 ng/uL
C0051 PCR product:22.8 ng/uL
B0015 PCR product:24.6 ng/uL

protocol:
total volume 20 uL

R0062(104bp) 1 uL
C0051(803bp) 8 uL
B0015(153bp) 1.5uL
ddH2O 4.85 uL
buffer 2uL
dNTP 0.4uL
Taq polymerase 0.25uL
MgCl2 2uL

PCR cycle including:

Preheat: 94 degree celcius, 5min
10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
(R0062-F primer 1uL;C0062-B0015-R(I) primer 1uL)to the remaining 15 uL PCR product.

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Result:
we don't get the product we want.We guess the templates are not the right products,maybe they have mutations .So we decide to do the overlap PCR of bateriaI to test whether the method is suitable.

2015 28/7

Plasmid Extraction of gRNA-AcrB and gRNA-EmrE

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Gel Extraction

Material including: R0061

Case 1: Sangon Biotech.

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Case 2: Axygen

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add 400~600 ul Buffer DE-A and then incubate the EP tubes in thermostat water bath about 75 degree centigrade within 10 mins.
After gel is all dissolved, add 1/3 volumn of Buffer DE-B into EP tubes. The solution will turn yellow, illustrating complete dissolved.
Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul Buffer W1 in a centrifuge at 9000Xg about 30s and pour out the solution.
Add 700 ul Buffer W2 in a centrifuge at 9000Xg about 30s and pour out the solution.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Overlapping Fragments

We conduct the overlapping again.
However, the sample has a little difference. The concentration of micF changes to 58.7ng/uL. According to our sample extracted from PCR and calculation, ingradients characterize as below:

Name	Concentration(ng/uL)	Length(bp)
micF	57.8	358
SoxS	27.5	705
C0061(LuxI)	18.5	656
B0015(Terminator)	9.5	143
Total		1161

The theoratical volumn proportion is:

SoxS-C0061-B0015: 5: 7: 3
micF-C0061-B0015: 1.3: 7: 3

Firstly, to ligate these parts, we need 10 cycles without primer:
for circuit SoxS-C0061-B0015:

Name	Volumn(uL)
SoxS	5
C0061	7
B0015	3
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
10X buffer with KCl	2
ddH2O	0.35

for circuit micF-C0061-B0015:

Name	Volumn(uL)
micF	1.3
C0061	7
B0015	3
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
10X buffer with KCl	2
ddH2O	4.1

PCR cycle including:

Preheat: 94 degree celcius, 5min
10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Primers are the same as before.

Name	Volumn(uL)
Template	5
Taq Polymerase	0.25
MgCl2	2
dNTP	0.4
micF-F(BB-SoxS-F)	1
B0015-R	1
10X buffer with KCl	2
ddH2O	8.35

15ul-protocol:

Name	Volumn(uL)
Solution	15
micF-F(BB-SoxS-F)	1
B0015-R	1
10X buffer with KCl	2
ddH2O	1

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

PCR of Cas9(80l)

material:

8 uL buffer with KCl
35 uL dd water
8 uL Plasmid with Cas9
4 uL primer R
4 uL primer F
8 uL MgCl2
8 uL dNTPs
5 uL DMSO
1 uL Tap polymerase

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

图片名称

2015 29/7

Negative Chemotaxis Characterization

Done by Wang Luyao
Term 1 results

I put the paper on this plate after the E.coli grows homogeneously， so let's see the result later.

the controlled plate

the last plate was contaminated

Term 2

material:

LB solid medium plates x6

0.8、0.6、0.4、0.2、0.1g/ml Chloramphenicol

E.coli Top10

filter paper

Enzyme Digestion

Performing twice separately

Both as following protocol:

Name	Volumn(uL)
pSB1C3	10
XbaI	1
SpeI	1
green buffer	2
ddH2O	6

Overlap PCR of bacteriaI

material:

micF PCR product :33.6ng/uL
C0061 PCR product: 18.5ng/uL
B0015 PCR product:9.2ng/uL
micF-F primer
B0015-R primer

protocol:
total volume: 25uL

Taq Master Mix 12.5uL
micF 2uL
C0061 6uL
B0015 2.6uL
ddH2O 1.9uL

PCR cycle including:

Preheat: 94 degree celcius, 5min
10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)
Elongation for whole at 72 degree celcius 10min
4 celcius degree for preserveation.

After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
to the remaining 15 uL PCR product.

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)
Elongation for whole at 72 degree celcius 10min
4 celcius degree for preserveation.

Results:
We don't get the product we want.We make some research and guess that the probable reasons are the following:Taq polymerase can cause 3'd A to PCR product;the annealing temperature is unsuitable;the length of overlap fragment isn't enough .

2015 30/7
Negative Chemotaxis Characterization

Done by Wang Luyao
7.30

Term 2 results

The 0.8、0.6、0.4 plates just look similar, the denser Chloramphenicol the paper hold， the bigger the clear zone is.

but the 0.2 and 0.1g/ml plates are different, Top10 grows denser on the edge of the clear zone, I supposed it can show that E.coli has moved. So next time I'll try under 0.2g/ml.

the controlled plate

PCR of C0062 and R0010

material:
- 10 ul Taq PCR Master Mix
- 7 ul ddH2O
- 1 ul Primer F
- 1 ul Primer R
- 1 ul C0062 or R0010
PCR cycle including:
1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

2015 31/7

Negative Chemotaxis Characterization

Done by Wang Luyao
Term 3, material:

LB solid medium plates x5
0.2、0.1、0.05、0.025g/ml Chloramphenicol
E.coli Top10
filter paper

PCR of micF, SoxS, C0061 and R0015

material(volume 50uL):

25 ul Taq PCR Master Mix
15 ul ddH2O
2.5 ul Primer F
2.5 ul Primer R
2.5 ul Top10 culture, Top10 culture, C0061 or R0015

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

PCR of tRNA

Primers	Sequence(5' to 3')
tRNA-F	GAATTCGCGGCCGCTTCTAGATAATACGACTCACTATAGGGAATACAAGCTACTTGTTCTTTTTGCAATGGACGAGTTCGAAATGATT
tRNA-R	CTGCAGCGGCCGCTACTAGTTTACAGACGTTTGCGAATTG
tRNA-F2	TTTAATCCCCGTCCACTGGGTAATACGACTCACTATAGGGAATAC

Protocol (volume 20uL):

2 ul Taq Buffer
0.4 ul dNTP
1 ul tRNA-F
1 ul tRNA-R
0.25 ul Taq DNA Polymerase
2 ul MgCl2
1 ul tRNA
12.35 ul ddH2O

Protocol (volume 20uL):

2 ul Taq Buffer
0.4 ul dNTP
1 ul tRNA-F2
1 ul tRNA-R
0.25 ul Taq DNA Polymerase
2 ul MgCl2
1 ul tRNA
12.35 ul ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
25 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min
4 celcius degree for preserveation.

Result
Only one sample get PCR product, concentration 441ng/μL. tRNA-F2 dosen't work for wrong template.
Remember to review gene map before experiments!

PCR of cheZ

material:
Top 10 Bacterial Solution

Primers produced from Sangon Biotech Co.,Ltd.

Primers	Sequence(5' to 3')
cheZ-F	GTGCAATTGATCCAGGAGCTCAGCCAGGC
cheZ-T7-R	GCTCCTGGATCAATTGCACATAAATTTTCTCCTCTTTTGCAAAAAGAA

for cheZ (~600bp)
Protocol as this:

2.5 μl Taq buffer with KCl
2.5 μl dNTPs
0.75 μl cheZ-F Primer
0.75 μl cheZ-T7-R Primer
0.5 μl Kod DNA Polymerase(600bp/min)
1.0 μl MgCl2
2 uL Top10 Solution
15 μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min 30s)
Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)
4 celcius degree for preserveation.

It turned out we used wrong primer:(

PCR of CheZ and B0015

material:

TOP 10 baterial solution
PAO1 bacterial solution
B0015 plasmid :93.0 ng/uL

protocol:
total system 25uL

buffer 2.5 uL
dNTPs 2.5uL
MgSO4 2uL
primer-F 0.75 uL
primer-R 0.75 uL
bacterial solution/plasmid 1uL
KOD-Plus enzyme 0.5uL
ddH2O 17uL

PCR cycle including:

Preheat: 94 degree celcius, 2min
35 cycles containing: degradation(94 degree celcius, 15s; annealing(55 degree celcius, 30s and elongation(68 degree celcius 50s)
Elongation for whole at 68 degree celcius 7min.
4 celcius degree for preserveation.

The protocol is from lab319,the enzyme is KOD-Plus.

Result:
We don't get the product we want .It turned out because of wrong primer we implemented.

Transformation of C0062

the protocol as following
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate

2015 1/8

Negative Chemotaxis Characterzation

Done by Wang Luyao
Term 3 results

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qqqq 034.JPG

this time I got the similar results, but I cannot tell whether the bacteria is moved or didn't grow up.

so let's see the term 1 result:

qqwqw 002.JPG

I put the paper on this plate after the E.coli grows homogeneously. Now after I took it out I got this.

It's obviously that the bacteria moved. If E.coli died, the medium cannot be clear, the bodies will stay. In the middle of the area, there was a bubble formed by the medium and the wet paper. There is dilute Chloramphenicol, so the some bacteria moved there.

So we qualitatively proved that E.coli negative chemotaxis !!!

PCR of cheZ

Material:
Top 10 Bacterial Solution

Primers produced from Sangon Biotech Co.,Ltd.

Primers	Sequence(5' to 3')
cheZ-F	GTGCAATTGATCCAGGAGCTCAGCCAGGC
che-Ter-R	TTTATTTGATGCCTGGCTCTAGTATCAGAAACCCAGGCTGGACA

for cheZ (~600bp)
Protocol as this:

5 μl Taq buffer with KCl
1 μl dNTPs
2.5 μl cheZ-F Primer
2.5 μl che-Ter-R Primer
0.65 μl Pfu DNA Polymerase(600bp/min)
5 μl MgCl2
2.5 uL Top10 Solution
31 μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min 30s)
1.Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

No results: May be because of Pfu enzyme has already been out of date.

Protocol again:

Ingredients	Volumn(uL)
2X Taq Master Mix	25
cheZ-F	2.5
che-Ter-R	2.5
Top10 Solution	5
ddH2O	15

Ingredients	Volumn(uL)
2X Taq Master Mix	25
cheZ-F	2.5
che-Ter-R	2.5
Top10 Solution	5
ddH2O	12.5
DMSO	2.5

No results：it turns out cheZ sequence in TOP10 is different from PAO1, we designed after PAO1

Protocol again:

Ingredients	Volumn(uL)
2X Taq Master Mix	25
cheZ-F	2.5
che-Ter-R	2.5
PAO1 Solution	5
ddH2O	15

Ingredients	Volumn(uL)
Taq buffer with KCl	5
dNTPs	1
che-Ter-R	2.5
PAO1 Solution	5
ddH2O	28.35
MgCl2	5
Taq Polymerase	0.65

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

PCR of tRNA

Protocol (volume 20uL):

2 ul Taq Buffer
0.4 ul dNTP
1 ul tRNA-F2
1 ul tRNA-R
0.25 ul Taq DNA Polymerase
2 ul MgCl2
1 ul tRNA
12.35 ul ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min
4 celcius degree for preserveation.

Gel Extraction of tRNA

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Add 1.5mL Buffer B2 and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 20uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 10 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Result
Concentration 64ng/ul.

PCR of CheZ

material:
Top 10 Bacterial Solution

protocol:
total system 25uL

buffer 2.5 uL
dNTPs 2.5uL
MgSO4 3uL
primer-F 0.75 uL
primer-R 0.75 uL
bacterial solution/plasmid 1uL
KOD-Plus-Neo enzyme 0.5uL
ddH2O 16uL

PCR cycle including:

Preheat: 94 degree celcius, 2min
35 cycles containing: degradation(98 degree celcius, 10s; annealing(55 degree celcius, 30s and elongation(68 degree celcius 30s)
Elongation for whole at 68 degree celcius 7min.
4 celcius degree for preserveation.
The protocol is from lab 319.

PCR of T7 promoter

Primers	Sequence(5' to 3')
T7-F	GAATTCGCGGCCGCTTCTAG
T7-R2	agacatgcaatttttttcattccgattttctcctcttttgcaaaaagaacaagtagct
T7 Promoter1	GAATTCGCGGCCGCTTCTAGAtaatacgactcactatagggaatacaagctacttgttctttttgca
T7 Promoter2	tgcaaaaagaacaagtagcttgtattccctatagtgagtcgtattaTCTAGAAGCGGCCGCGAATTC

Protocol (volume 20uL):

10 ul Mix
1 ul T7-F
1 ul T7-R2
1 ul T7 Promoter1
1 ul T7 promoter2
6 ul ddH2O

PCR cycle including:

35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 30s)
Elongation for whole at 72 degree celcius 7min.
4 celcius degree for preserveation.

PCR Purification of T7

Add Buffer B3 in 5 times volumn of PCR product.
Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and measure the concentration of DNA and A260/A280 for further study.

Result
Concentration 28ng/ul.

PCR of T7 promoter

Conduct the PCR again.

Protocol (volume 20uL):

10 ul Mix
1 ul T7-F
1 ul T7-R2
1 ul T7 Promoter1
1 ul T7 promoter2
6 ul ddH2O

PCR cycle including:

35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 30s)
Elongation for whole at 72 degree celcius 7min.
4 celcius degree for preserveation.

Seamless Clone of tRNA Plasmid

Protocol:

8.5 ul Seamless cloning Master Mix
3 ul linear PSB1C3 plasmid
2 ul tRNA
6.5 ul ddH2O
Incubate at 50℃ for 60min.

Seamless Clone of Anchor Plasmid

Protocol:

8.5 ul Seamless cloning Master Mix
3 ul linear PSB1C3 plasmid
1 ul T7 promoter
1 ul COMPX
6.5 ul ddH2O
Incubate at 50℃ for 60min.

Transformation of tRNA Plasmid & Anchor Plasmid

Procedure:

Add 4ul clone product to competent cells, mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
4000rpm centrifuge for 3min, discard 500ul supernate.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

2015 2/8

Plasmid Extraction of C0061

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

PCR of cheZ and OprF from PAO1

Yesterday, we may extract cheZ from PAO 1, though with a lot of primer dimer. Today, DMSO will be implemented trying to eliminate these fragements.

Ingredients	Volumn(uL)
Taq buffer with KCl	5
dNTPs	1
cheZ-F	2.5
cheZ-Ter-R	2.5
PAO1 Solution	5
ddH2O	25.85
MgCl2	5
Pfu Polymerase	0.65
DMSO	2.5

Ingredients	Volumn(uL)
Taq buffer with KCl	5
dNTPs	1
Opr-F	2.5
OprF-Ter-R	2.5
PAO1 Solution	5
ddH2O	25.85
MgCl2	5
Pfu Polymerase	0.65
DMSO	2.5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min 30 s)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

PCR Purification of T7

Add Buffer B3 in 5 times volumn of PCR product.
Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Gel Extraction

Material including: OprF, cheZ

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Colonial PCR of PSB1C3-T7-COMPX & PSB1C3-T7-tRNA

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R or tRNA-R Primer
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

No Result.

Colonial PCR of PSB1C3-T7-COMPX & PSB1C3-T7-tRNA

Conduct the PCR again.

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R or tRNA-R Primer
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

No Result.

PCR Purification of tRNA & COMPX

Add Buffer B3 in 5 times volumn of PCR product.
Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and measure the concentration of DNA and A260/A280 for further study.

Result
tRNA 26ng/ul, COMPX 24ng/ul.

Transformation of tRNA Plasmid & Anchor Plasmid

Procedure:

Add 4ul clone product to competent cells, mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
4000rpm centrifuge for 3min, discard 500ul supernate.
Spread half of the bacteria culture on plate with chloramphenicol, incubate overnight at 37℃.

2015 3/8

Colonial PCR of PSB1C3-T7-COMPX & PSB1C3-T7-tRNA

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R or tRNA-R Primer
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

PCR of OprF from PAO1

Yesterday, we may extract cheZ from PAO 1, though with a lot of primer dimer. Today, DMSO will be implemented trying to eliminate these fragements.

Ingredients	Volumn(uL)
Taq buffer with KCl	5
dNTPs	1
Opr-F	2.5
OprF-Ter-R	2.5
PAO1 Solution	5
ddH2O	28.35
MgCl2	5
Pfu Polymerase	0.65
DMSO	2.5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

PCR of C0061

material(volume 100uL):
• 6 ul MgCl2
• 71 ul ddH2O
• 4 ul Primer F
• 4 ul Primer R
• 2 ul dNTP
• 1 ul Taq DNA polymerase
• 10 ul 10*PCR Buffer
• 2 ul C0061
PCR cycle including:
1 Preheat: 94 degree celcius, 5min
2 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3 Elongation for whole at 72 degree celcius 1min
4 4 celcius degree for preserveation.

Gel Extraction

Material including: R0061
Preparation:
• Check out whether ethyl alcohol is added into Wash Solution.
• Check out whether Buffer B2 has sediment.
• Adjust the thermostat water bath at 50 degree centigrade.
Procedure:
5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
6 Measure out the weight of tubes of gel.
7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
11 Do the step 5 again.
12 Centrifuge the empty columns at 9000xg about 1min.
13 Open the columns and air them about 10 mins.
14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.

PCR for gRNA-AcrB and gRNA-EmrE

material:
gRNA-AcrB plasmid(280.7 ng/uL)
gRNA-EmrE plasmid(222.4 ng/uL)

PCR protocol:

For gRNA-AcrB and gRNA-EmrE, primers including gRNA-F, gRNA-R, protocol as following:

10μl Taq Master Mix
1μl gRNA-F Primer
1μl gRNA-R Primer
0.25μl Taq DNA Polymerase
6.75μl ddH2O
1μl gRNA-AcrB(or gRNA-EmrE) Plasmid

For gRNA, Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
gRNA-F	GCGTCTAGATTGACAGCTAGCTCAGTAGGTATAAT
gRNA-R	TAGTAGGTCGTATTAAAAAAAAGCACCGAC

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Gel Electrophoresis Characterization

gRNA-AcrB and gRNA-EmrE
图片名称

Gel Extraction of gRNA-AcrB and gRNA-EmrE

Material including: gRNA-AcrB and gRNA-EmrE

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

the concentration as following:
gRNA-AcrB: 44 ng/uL
gRNA-EmrE: 41 ng/uL

PCR for T7-tet Promoter

PCR protocol:

For T7-tet promoter, primers including T7-tet-F, T7-tet-R, protocol as following:

10μl Taq Master Mix
1μl T7-tet-F Primer
1μl T7-tet-R Primer
1μl template-F Primer
1μl template-R Primer
0.25μl Taq DNA Polymerase
5.75μl ddH2O

Use template-F Primer and template-R Primer as templates

For T7-tet, Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
template-F	taatacgactcactatgatagaaaagaggagaaaatc
template-R	gattttctcctcttttctatcatagtgagtcgtatta
T7-tet-F	Ttaatacgactcactatgatagaaaagaggag
T7-tet-R	gtatttcttatccattccgattttctcctcttttctat

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Gel Electrophoresis Characterization

T7-tet Promoter
图片名称

Gel Extraction of T7-tet Promoter

Material including: T7-tet Promoter

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

the concentration as following:
T7-tet: 30 ng/uL

Seamless Clone of cheZ Plasmid and Circuit Containing E0040

Protocol:

Fragements	Length(bp)	Mass Required(ug)
linear PSB1C3 plasmid	2070	41.4
T7 Promoter	46	9.2
cheZ	683	13.66
B0015	143	2.86

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	3 ul
T7 Promoter	1ul(+3 folds ddH2O)
cheZ	1ul(+1 fold ddH2O)
B0015	1ul(+9 folds ddH2O)
ddH2O	5.5ul

Incubate at 50℃ for 60min.

Fragements	Length(bp)	Mass Required(ug)
linear PSB1C3 plasmid	2070	41.4
R0061	30	0.6
E0040	720	14.4
B0015	143	2.86

Ingredients	Volumn(uL)
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	3 ul
R0061	1ul(+20 fold ddH2O)
E0040	1ul(+1 fold ddH2O)
B0015	1ul(+9 folds ddH2O)
ddH2O	5.5ul

PCR for Cas9-PART I and Cas9-PART II

material:
Cas9 plasmid(511.3 ng/uL)

PCR protocol:

For Cas9-PART I and Cas9-PART II, primers including Cas9-1-F, Cas9-1-R,Cas9-2-F, Cas9-2-R, protocol as following:

10μl Taq Master Mix
1μl Cas9-1-F(or Cas9-2-F) Primer
1μl Cas9-1-R(or Cas9-2-R) Primer
0.25μl Taq DNA Polymerase
6.75μl ddH2O
1μl Cas9 Plasmid

For Cas9, Primers produced from Sangon Biotech Co.,Ltd.

Primer	Sequence(5'-3')
Cas9-1-F	ggaatggataagaaatactcaataggcttag
Cas9-1-R	gctgtttcatcaccttatcatcaaaga
Cas9-2-F	gataaggtgatgaaacagcttaaacgtc
Cas9-2-R	ctactagtgggaccattcaaaacagcatagc

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5min)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Gel Electrophoresis Characterization

Cas9-PART I and Cas9-PART II
图片名称

Gel Extraction of Cas9-PART I and Cas9-PART II

Material including: Cas9-PART I and Cas9-PART II

Preparation:

Check out whether ethyl alcohol is added into Wash Solution.
Check out whether Buffer B2 has sediment.
Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
Measure out the weight of tubes of gel.
Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
Do the step 5 again.
Centrifuge the empty columns at 9000xg about 1min.
Open the columns and air them about 10 mins.
Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

the concentration as following:
Cas9-PART I : 31 ng/uL
Cas9-PART II: 16 ng/uL

Seamless Clone of tRNA Plasmid

Protocol:

8.5 ul Seamless cloning Master Mix
3 ul linear PSB1C3 plasmid
1 ul tRNA
7.5 ul ddH2O

Incubate at 50℃ for 60min.

Seamless Clone of Anchor Plasmid

Protocol:

8.5 ul Seamless cloning Master Mix
3 ul linear PSB1C3 plasmid
1 ul T7 promoter
1 ul COMPX
6.5 ul ddH2O

Incubate at 50℃ for 60min.

Transformation of tRNA Plasmid & Anchor Plasmid

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

2015 4/8

PCR for micF and C0061

Protocol:

Ingradients	Volumn(uL)
10X PCR buffer	8
dNTPs	1.6
micF-F	4
micF-luxI-R	4
Top10 Solution	8
Pfu Polymerase	0.5
ddH2O	53.9

Ingradients	Volumn(uL)
10X PCR buffer	8
dNTPs	1.6
C0061-F	4
C0061-B0015-R	4
C0061	8
Pfu Polymerase	0.5
ddH2O	53.9

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30s)
Elongation for whole at 72 degree celcius 7 min
4 celcius degree for preserveation.

Seamless Clone of circuit I

material:
pSB1C3(XbaI&SpeI digest) 2062bp 7.1ng/uL
micF 402bp 58.7ng/uL
C0061 656bp 27.4ng/uL
B0015 142bp 7.1ng/uL
seamless clone kit

protocol:
pSB1C3 12.4uL
micF 0.5uL
C0061 1.73uL
B0015 1.46uL
seamless clone mix 8.5uL

Incubate at 50℃ for 60min.

Transformation of circuit I

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 3min.
Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

2015 5/8

Seamless Clone of circuit I

material:
pSB1C3(XbaI&SpeI digest) 2062bp 7.1ng/uL
micF 402bp 58.7ng/uL
C0061 656bp 27.4ng/uL
B0015 142bp 7.1ng/uL
seamless clone kit

protocol:
pSB1C3 12.4uL
micF 0.5uL
C0061 1.73uL
B0015 1.46uL
seamless clone mix 8.5uL

Incubate at 50℃ for 60min.

Transformation of circuit I

Protocol:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 3min.
Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

Transformation of pSB1C3

protocol:

Add 2ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 3min.
Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

Double digest of pSB1C3

protocol:
pSB1C3 plasmid solution 10ul
XbaI 1ul
SpeI 1ul
Tango.buffer 2ul
ddH2O 6ul

incubate at 37℃ for 2 hours.

2015 6/8

Colonial PCR of PSB1C3-T7-COMPX & PSB1C3-T7-tRNA

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R or tRNA-R Primer
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

Colonial PCR of PSB1C3-T7-cheZ & PSB1C3-R0051-E0040-B0015

Protocol(volume 13.5ul)

0.25μl Primer 1
0.25μl Primer 2
6.5μl Mix
1μl Bacteria Clone
5.5μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

Overlap Circuit of gRNA-Cas9

图片名称

Overlapping Fragments

In order to ligate the fragements gRNA-ArcB or gRNA-EmrE, T7-tet, Cas9-PART I and Cas9-PART II, we need to make the equal mole proportion.

The mole of DNA molecule is calculated in the following ways:

图片名称

among which, c is the concentration of DNA, V is the volumn required and n is the length of DNA. Because of the same molar mass of As, Gs, Cs and Ts, computation of same molar mass is omitted.

According to our sample extracted from PCR and calculation, ingradients characterize as below:

Name	Concentration(ng/uL)	Length(bp)
gRNA-ArcB	44	162
gRNA-EmrE	41	162
T7-tet	30	56
Cas9-PART I	31	1954
Cas9-PART II	16	2358
Total		4530

The theoratical volumn proportion is:

gRNA-ArcB-T7-tet-Cas9-PART I-Cas9-PART II : 2: 1: 31: 64
gRNA-EmrE-T7-tet-Cas9-PART I-Cas9-PART II: 2: 1: 31: 64

Firstly, to ligate these parts, we need 10 cycles without primer:
for circuit gRNA-ArcB-T7-tet-Cas9-PART I-Cas9-PART II:

Name	Volumn(uL)
gRNA-ArcB	0.2
T7-tet	0.1
Cas9-PART I	3.1
Cas9-PART II	6.4
Taq Polymerase	0.25
dNTP(+MgCl2)	2
10X buffer	2
DMSO	1
ddH2O	4.95

for circuit gRNA-EmrE-T7-tet-Cas9-PART I-Cas9-PART II:

Name	Volumn(uL)
gRNA-EmrE	0.2
T7-tet	0.1
Cas9-PART I	3.1
Cas9-PART II	6.4
Taq Polymerase	0.25
dNTP(+MgCl2)	2
10X buffer	2
DMSO	1.25
ddH2O	4.7

PCR cycle including:

Preheat: 94 degree celcius, 5min
10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 6.5min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Then, we extracted 5 ul as template, and 15 ul adding with primer:
Primers were synthesized from Sangon Biotech. Co.,Ltd.

Primers	Sequence(5' to 3')
gRNA-F	GCGTCTAGATTGACAGCTAGCTCAGTAGGTATAAT
Cas9-2-R	CTACTAATGGGACCATTCAAAACAGCATAGC

5ul-template protocol:

Name	Volumn(uL)
Template	5
Taq Polymerase	0.25
dNTP(+MgCl2)	2
10X buffer	2
DMSO	1.25
ddH2O	7.5
gRNA-F	1
Cas9-2-R	1

15ul-protocol:

Name	Volumn(uL)
Solution	15
gRNA-F	1
Cas9-2-R	1
DMSO	1
10X buffer	2

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 6.5min)
Elongation for whole at 72 degree celcius 1min
4 celcius degree for preserveation.

Aug 6th, 2015

Plasmid Extraction of Circuit 1, B0015, T7

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Enzyme Digestion

Protocol:
Name Volumn(uL)
pSB1C3 10
XbaI 1
SpeI 1
10* Buffer Tango 2
ddH2O 15

Colonial PCR of PSB1C3-T7-COMPX

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

Double Digest of T7 Plasmid

Protocol (Volume 20ul)

4ul Tango Buffer
2ul BamHI
2ul SalI
10ul T7 Plasmid
2ul ddH2O
Incubate at 37℃ for 3h.

Colonial PCR of PSB1C3-T7-COMPX

Conduct the PCR again.

Protocol(volume 20ul)

1μl T7-F Primer
1μl COMPX-R
10μl Mix
1μl Bacteria Clone
7μl ddH2O

PCR cycle including:

Preheat: 94 degree celcius, 7min
35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)
Elongation for whole at 72 degree celcius 10min.
4 celcius degree for preserveation.

Double Digest of T7 Plasmid

Protocol (Volume 20ul)

4ul Tango Buffer
2ul BamHI
2ul SalI
10ul T7 Plasmid
2ul ddH2O
Incubate at 37℃ overnight.

2015 10/8

PCR of C0062

material(volume 100uL):
• 6 ul MgCl2
• 71 ul ddH2O
• 4 ul Primer F
• 4 ul Primer R
• 2 ul dNTP
• 1 ul pfu DNA polymerase
• 10 ul 10*PCR Buffer
• 2 ul C0062
PCR cycle including:
1 Preheat: 94 degree celcius, 5min
2 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3 Elongation for whole at 72 degree celcius 1min
4 4 celcius degree for preserveation.

2015 11/8

Seamless Clone of pSB1C3-R0010-C0062-B0015

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	2 ul
R0010	0.4ul
C0062	0.6ul
B0015	2.4ul
ddH2O	6.1ul

Incubate at 50 degree celcius 1 h.

Seamless Clone of pSB1C3-T7-COMPX

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	2ul
T7	2ul
COMPX	1ul
ddH2O	6.5ul

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	2ul
T7	1ul
COMPX	1ul
ddH2O	14.5ul

Incubate at 50 degree celcius 1 h.

Transformation of pSB1C3-T7-COMPX

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

Enzyme Digestion of pSB1C3-T7-COMPX and pSB1C3-tRNA

Ingredients	Volumn
Tango Buffer	2ul
plasmid	10ul
Spe I	1ul
Xba I	1ul
ddH2O	6ul

Incubate at 37 degree celcius for 2 h.

2015 12/8

Transformation of pSB1C3-R0010-C0062-B0015

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

Transformation of pSB3A3

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
Spread on plate with Ampcilin, incubate overnight at 37℃.

Plasmid Extraction of T7

Procedure including:
1.Absorb 4.5 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

PCR of OprF from PAO1

Ingredients	Volumn(uL)
2XTaq Mix	25
OprF-F	2.5
OprF-Ter-R	2.5
PAO1	2.5
ddH2O	17.5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

2015 13/8

Plasmid Extraction of R0062, B0015, SCVE, T7

Procedure including:
1.Absorb 4.5 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

PCR of R0062, C0051, E0040, B0015

Ingredients	Volumn(uL)
PCR Buffer	5
Primer 1	2.5
Primer 2	2.5
Plasimid Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

Pre-annealing of R0051

Ingredients	Volumn(uL)
R0051-F1	10
R0051-R1	10

Incubate solution in 95 degree celcius 30 min and then used for PCR.

PCR of R0051, T7-OprF, T7-SCVE, SCVE, micF and soxS

Ingredients	Volumn(uL)
PCR Buffer	5
Primer 1	2.5
Primer 2	2.5
Plasimid Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

PCR of OprF, micF, SoxS

Ingredients	Volumn(uL)
PCR Buffer	5
Primer 1	2.5
Primer 2	2.5
Bacterial Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

Gel Extraction

Material including: R0061, E0040, B0015, T7-OprF
Preparation:
• Check out whether ethyl alcohol is added into Wash Solution.
• Check out whether Buffer B2 has sediment.
• Adjust the thermostat water bath at 50 degree centigrade.

Procedure:
5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
6 Measure out the weight of tubes of gel.
7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
11 Do the step 5 again.
12 Centrifuge the empty columns at 9000xg about 1min.
13 Open the columns and air them about 10 mins.
14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.

PCR of OprF, SoxS, micF

Ingredients	Volumn(uL)
PCR Buffer	5
Primer 1	2.5
Primer 2	2.5
Bactieral Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(Temperature gradient: 48 degree celcius, 50 degree celcius, 53 degree celcius and 55 degree celcius 30s) and elongation(72 degree celcius 2min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

Gel Extraction

Material including: T7-SCVE, SCVE
Preparation:
• Check out whether ethyl alcohol is added into Wash Solution.
• Check out whether Buffer B2 has sediment.
• Adjust the thermostat water bath at 50 degree centigrade.

Procedure:
5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
6 Measure out the weight of tubes of gel.
7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
11 Do the step 5 again.
12 Centrifuge the empty columns at 9000xg about 1min.
13 Open the columns and air them about 10 mins.
14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min

Enzyme Digestion of pSB1C3

incubate at 37 degree celcius about 2 h

Ingredients	Volumn(uL)
Enzyme 1	1
Enzyme 2	1
pSB1C3	4
Tango Buffer	4
ddH2O	10

strategy	Enzyme 1	Enzyme 2
Strategy 1	EcoRI	PstI
Strategy 2	EcoRI	SpeI
Strategy 1	XbaI	PstI
Strategy 2	XbaI	SpeI

2015 14/8

PCR of C0061, micF/SoxS-binding C0061

for C0061

Ingredients	Volumn(uL)
PCR Buffer	5
C0061-F	2.5
C0061-B0015-RI	2.5
Plasimid Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

for micF-C0061

Ingredients	Volumn(uL)
PCR Buffer	5
micF-R-BB	2.5
B0034-C0061-R	2.5
Plasimid Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

for SoxS-C0061

Ingredients	Volumn(uL)
PCR Buffer	5
SoxS-R-BB	2.5
B0034-C0061-R	2.5
Plasimid Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2min)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

Seamless ligation of pSB1C3-T7-SCVE-B0015

Ingradients	Volumn(uL)
pSB1C3	1.2
T7-SCVE	1
SCVE	1
B0015	1
Seamless Cloning mix	8.5
ddH2O	to 20

Incubate at 50 degree celcuis 1 h

2015 15/8

PCR of OprF, SoxS

Ingredients	Volumn(uL)
PCR Buffer	5
Primer 1	2.5
Primer 2	2.5
Bacterial Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

PCR of micF-C0061

Ingredients	Volumn(uL)
PCR Buffer	5
micF-F-BB	2.5
B0034-C0061-R	2.5
Bacterial Solution	2.5
ddH2O	31.9
Pfu Polymerase	0.6
dNTP mix	5

PCR cycle including:

Preheat: 94 degree celcius, 5min
35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)
Elongation for whole at 72 degree celcius 10 mins
4 celcius degree for preserveation.

Gel Extraction

Material including: micF, pSB1C3
Preparation:
• Check out whether ethyl alcohol is added into Wash Solution.
• Check out whether Buffer B2 has sediment.
• Adjust the thermostat water bath at 50 degree centigrade.

Procedure:
5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
6 Measure out the weight of tubes of gel.
7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
11 Do the step 5 again.
12 Centrifuge the empty columns at 9000xg about 1min.
13 Open the columns and air them about 10 mins.
14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.

Ligation of pSB1C3-tRNA

incubate at 16 degree celcius more than 20 h

Ingredients	Volumn(uL)
T4 DNA ligation Buffer	2
T4 DNA ligase	1
pSB1C3 digested by EcoRI and PstI	1
tRNA	6
ddH2O	11

Seamless Cloning of pSB1C3-micF-C0061-B0015

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	2 ul
micF	1ul
C0061	1ul
B0015	1ul
ddH2O	6.5ul

Incubate at 50℃ for 60min.

Seamless Cloning of pSB1C3-T7-COMPX

Ingredients	Volumn
Seamless cloning Master Mix	8.5 ul
linear PSB1C3 plasmid	2 ul
COMPX	1ul
ddH2O	8.5ul

Incubate at 50℃ for 60min.

Transformation of pSB1C3-micF-C0061-B0015 and pSB1C3-T7-COMPX

Procedure:

Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.
Heat in 42℃ bath for 90s. Then put on ice for 5min.
Add 500ul SOC culture, incubate at 37℃ for 45min.
Spread on plate with chloramphenicol, incubate overnight at 37℃.

Raw-data

Raw-data

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+	<li><a href="https://2015.igem.org/Team:USTC/Achievements" class="waves-effect waves-light">Achivements</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Safety" class="waves-effect waves-light">Safety</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Safety" class="waves-effect waves-light">Safety</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Practices" class="waves-effect waves-light">Policy&Practices</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Practices" class="waves-effect waves-light">Policy&Practices</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Collaborations" class="waves-effect waves-light">Collaborations</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Collaborations" class="waves-effect waves-light">Collaborations</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Team" class="waves-effect waves-light">Team</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Team" class="waves-effect waves-light">Team</a></li>
-  <li><a href="https://2015.igem.org/Team:USTC/Attributions" class="waves-effect waves-light">Attributions</a></li>
+	<li><a href="https://2015.igem.org/Team:USTC/Attributions" class="waves-effect waves-light">Attributions</a></li>
 </ul>
+<div class="row">
+  <div class="col offset-m1 offset-l2 s12 m10 l8">
+    <div id="Protocols" class="row">
+      <div class="card hoverable">
+        <div class="col s12 m9">
+        	<div class="card-content">
+          	Protocols
+          </div>
+        </div>
+        <div class="col hide-on-small-only m3">
+          <div class="toc-wrapper pinned">
+            <ul class="section table-of-contents">
+              <li>
+                <a href="#Protocols">Protocols</a>
+              </li>
+            </ul>
+          </div>
+        </div>
+      </div>
+    </div>
-<h2>Notebook</h2>
+    <div id="Daily-Notes-Stage-1" class="row">
+      <div class="card hoverable">
+        <div class="col s12">
+        	<div class="card-content">
+          	<ul class="cbp_tmtimeline">
+          		<li>
+                <time class="cbp_tmtime" datetime="2015-07-04 00:00"><span>2015</span> <span>4/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Medium preparation</h5>
+                  <p>Prepare and keep a part of solid medium and broth in the refrigerator as a backup.</p>
+                  <h5>Cell culture</h5>
+                  <p>Culture the cell,TOP10, with LB liquid solution overnight.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-05 0:0"><span>2015</span> <span>5/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Design protocol</h5>
+                  <ol>
+                    <li>Take the solid medium, heat it until liquid. Then make two plate of solid medium, evenly. Cool it to the room temperature.</li>
+                    <li>prepare antibiotic solution with concentration gradient
+                      <br>a. prepare antibiotic(neomycin) solution with ddH2O at 50mg/ml as mother solution 1. And dilute it to 10% concertration(5mg/ml) with ddH2O as mother solution 2. Then prepare 8 parts of solution as this:</li>
+                  </ol>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Number</th>
+                        <th>Solution 1(V/uL)</th>
+                        <th>Solution 2(V/uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>0</td>
+                        <td>0</td>
+                        <td>0</td>
+                      </tr>
+                      <tr>
+                        <td>1</td>
+                        <td>0</td>
+                        <td>0.2</td>
+                      </tr>
+                      <tr>
+                        <td>2</td>
+                        <td>0</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>3</td>
+                        <td>0.2</td>
+                        <td>0</td>
+                      </tr>
+                      <tr>
+                        <td>4</td>
+                        <td>0.4</td>
+                        <td>0</td>
+                      </tr>
+                      <tr>
+                        <td>5</td>
+                        <td>0.6</td>
+                        <td>0</td>
+                      </tr>
+                      <tr>
+                        <td>6</td>
+                        <td>0.8</td>
+                        <td>0</td>
+                      </tr>
+                      <tr>
+                        <td>7</td>
+                        <td>1</td>
+                        <td>0</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p> Keep them in the 70℃ water bath. Then add 800uL liquid solid medium into each Ep tubes. Transfer them into 50℃ water bath. </p>
+                  <ol>
+                    <li>Cut 4 hole in each plate of solid medium, whose diameter is at 8mm. </li>
+                    <li>Take 5mL culture into the 2 Ep tubes. Centrifuge it at twice. Then add liquid solid medium at 50℃, each 800uL, dissolve evenly. </li>
+                    <li>Take 200uL top10 solution into 8 Ep tubes each at 50℃. Then add the mixed solution into the hole at once until the hole is added up.</li>
+                  </ol>
+                  <p>Results: the raw results showed differerent characteristics of chemotaxis based on distinct antibiotic concentration.
+                    <br><img src="http://chuantu.biz/t2/10/1436687140x-1376436664.jpg" alt="0-3">
+                    <br><img src="http://chuantu.biz/t2/10/1436687142x-1376436670.jpg" alt="4-7"></p>
+                  <p>Analysis: 其中3号有最明显的趋化结果，说明这个趋化结果并不是完全正相关或负相关的。但是结论可能是存疑的，过量的加入固体培养基会导致可能的菌液溢出从而形成如此的趋化形状。因此实验配方需要进行更改。</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-06 00:00"><span>2015</span> <span>6/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <p>
+                    今天在延续着昨天的实验思路，利用灭菌的1mL枪头打洞，然后将含抗培养基和菌液混合，打入洞中，待凝固后放入恒温箱培养。
+                    <br>实验结果:这次并没有长出相应的环带。有可能是由于在放入之前被紫外灯照射了的缘故导致的细菌死亡。但是同时也有可能是由于孔洞较深，使得大肠杆菌没有办法从边缘逃逸。
+                  </p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-07 00:00"><span>2015</span> <span>7/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <p>前一天的培养基里头出现了<em>E.coli</em>,但是伴随着杂菌。
+                    <br>于是重复了7月6日的实验，此次打洞是，为了方便控制液体体积，这会使用的是灭菌的200uL枪头打洞。不过由于体积过小，导致仍有一部分液体溢出。</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-08 00:00"><span>2015</span> <span>8/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <p>发现7月7日的结果与7月5日结果有差别，并且各孔的趋化效果呈振荡形式，并且环的效果不是很明显。</p>
+                  <p>analyse：形成环的一部分孔洞是存在着溢出情况的，某种程度上印证的之前关于溢出的猜测。
+                    <br>可能E.coli的运动能力不是很强，引起环的明显程度不够，或者是由于抗生素的浓度过高，而杀死或抑制了细菌。后面实验还需要重新审核实验方案，并且需要克服由于加样无法精确掌握体积的问题。</p>
+                  <p>接下来的思路：可以考虑利用含有抗性的菌来做趋化实验，但是首先要求该种抗性仅仅是抗生素作用靶点的改变，而不是分解或者是抗生素失活的方式，以免改变抗生素的局部浓度。</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-09 00:00"><span>2015</span> <span>9/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>转化</h5>
+                  <p>今天转化的是来自刘玉婷实验室的TG1感受态细胞，感受态细胞是由刘玉婷与张启钧提前完成的，主要目的是为了检测：</p>
+                  <ul>
+                    <li>感受态细胞是否高效</li>
+                    <li>感受态细胞是否污染</li>
+                  </ul>
+                  <p>实验流程：</p>
+                  <ol>
+                    <li>冰上放置感受态细胞2~3min，一共4管</li>
+                    <li>加入5ul检测质粒，其中3管有100ul感受态细胞两支，200ul感受态细胞两支，其中100ul的感受态一支加入5ul质粒，一支加入1ul质粒，在冰上放置30min</li>
+                    <li>在42℃水浴锅中热激45s</li>
+                    <li>冰上放置3min</li>
+                    <li>加入900ulLB培养基培养55min</li>
+                    <li>离心12000g 15s，放弃上清液，加入200ulLB培养基</li>
+                    <li>涂板，板带有氨苄抗性</li>
+                    <li>培养20h观察实验结果</li>
+                  </ol>
+                  <h5>绿脓杆菌接菌</h5>
+                  <p>从金范老师那里拿来的绿脓杆菌生长完整，选取了其中的三个菌落挑单，发现绿脓杆菌单菌落相比大肠杆菌较大，培养基有异味。取5mL LB培养基直接将挑好的细菌用枪头打在其中培养16h次日以备使用。</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-10 00:00"><span>2015</span> <span>10/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>冻存绿脓杆菌</h5>
+                  <p>绿脓杆菌需要冻存，冻存的最后甘油体积分数为15%</p>
+                  <p>灭菌时配好的50%甘油进行灭菌，因为根据小木虫的经验帖，100%体积的甘油不宜直接灭菌</p>
+                  <p>甘油的密度为：1.26g/mL</p>
+                  <p>50%甘油（50mL）配方为：</p>
+                  <ul>
+                    <li>25mL 蒸馏水</li>
+                    <li>25mL（31.5g）甘油</li>
+                  </ul>
+                  <p>混匀之后即可灭菌，冻存管同时灭菌</p>
+                  <p>15%冻存甘油管的配方为（1mL）：</p>
+                  <ul>
+                    <li>700ul 培养菌液</li>
+                    <li>300ul 15%甘油溶液</li>
+                  </ul>
+                  <p>冻存于-40℃冰箱中，备用</p>
+                  <h5>涂板绿脓杆菌</h5>
+                  <p>早上：抽取培养过的绿脓杆菌取100ul加入LB固体培养基中进行涂板</p>
+                  <p>结果发现，细菌密度过浓，没有单菌落形成</p>
+                  <p>晚上：抽取培养的绿脓杆菌，进行1：4的培养基稀释，之后取100ul加入到LB固体培养基中涂板，过夜培养</p>
+                  <h5>转化</h5>
+                  <p>转化材料是来自洪炯老师的XL21 Gold感受态细胞，和昨日刘玉婷制备的TG1感受态细胞，希望进一步证明感受态细胞没有被污染，以及扩增pSBC1C3质粒，和T7启动子，tRNA part</p>
+                  <p>实验流程：</p>
+                  <ol>
+                    <li>冰上放置感受态细胞3min，一共4管</li>
+                    <li>稀释parts（T7启动子：2013年Kit5的6N孔，tRNA：2015年Kit5的21I孔），加入10ul灭菌过的蒸馏水，稀释混匀。</li>
+                    <li>加入1ul检测T7启动子与tRNA的parts至XL21 gold感受态细胞，加入5ulpSB1C3至XL21 Gold感受态细胞，在冰上放置30min</li>
+                    <li>在42℃水浴锅中热激55s</li>
+                    <li>冰上放置3min</li>
+                    <li>加入到5mL的氯霉素抗性的液体培养基。</li>
+                    <li>培养12h明日观察结果（21：00完成）</li>
+                  </ol>
+                  <h5>1st Colonial PCR for COMPX</h5>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl COMPX-F Primer</li>
+                    <li>1μl COMPX-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl TOP10 Solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 45℃, extend for 1 min.</p>
+                  <h5>1st Electrophoresis for Colonical PCR</h5>
+                  <p>No band except marker resulted due to Gelred past due.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-11 00:00"><span>2015</span> <span>11/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>重新转化实验</h5>
+                  <p>氯霉素的浓度昨天有误，今天重新进行配置，再养菌</p>
+                  <h5>重新制备感受态细胞</h5>
+                  <p>感受态细胞用Amp进行空白测试</p>
+                  <h5>重新对绿脓杆菌涂板</h5>
+                  <p>涂板稀释浓度增加到1:10与1:15，因为绿脓杆菌浓度过高，单菌落太少，不宜储存</p>
+                  <h5>2nd Colonial PCR for COMPX</h5>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl COMPX-F Primer</li>
+                    <li>1μl COMPX-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl TOP10 Solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation temperature varies from 50℃ to 60℃, extend for 1 min.</p>
+                  <h5>2nd Electrophoresis for Colonical PCR</h5>
+                  <p>Add Gelred in loading buffer. The band of PCR product appears in all samples.</p>
+                  <p>结果如下图所示：
+                    <br><img src="http://chuantu.biz/t2/10/1436619801x-1376436670.jpg" alt=""></p>
+                  <p>意外出现问题，由于胶用水去溶解了，导致Marker都没有跑开</p>
+                  <p><img src="http://chuantu.biz/t2/10/1436619751x-1376436664.jpg" alt=""></p>
+                  <p>该结果则较好的显示出了菌落PCR成功的p出了COMPX蛋白</p>
+                  <p>教训：</p>
+                  <ul>
+                    <li>误用琼脂</li>
+                    <li>误用水区溶解胶</li>
+                    <li>电压需要调整回90V，30min</li>
+                  </ul>
+                  <h5>3rd Colonial PCR for COMPX</h5>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl COMPX-F Primer</li>
+                    <li>1μl COMPX-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl TOP10 Solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 55℃, extend for 1 min.</p>
+                  <h5>3rd Electrophoresis for Colonical PCR</h5>
+                  <p>No results again.</p>
+                  <h5>1st Transformation</h5>
+                  <p>1 Add 1μl BBa K1492002 or 5μl pSB1C3 plasmid to competent cells.</p>
+                  <p>2 Heat at 42℃ for 45 s by mistake.</p>
+                  <p>3 Incubate on ice for 30 min.</p>
+                  <p>4 Heat at 42℃ for 45 s.</p>
+                  <p>5 Incubate on ice for 3 min.</p>
+                  <p>6 Add 900 μl LB medium, incubate at 37℃ for 1 h.</p>
+                  <p>7 Centrifuge and discard 85 μl LB medium.</p>
+                  <p>8 Resuspend cells and add 100 μl to LB medium with chloramphenicol. Incubate at 37℃. </p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-12 00:00"><span>2015</span> <span>12/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>4th Colonial PCR for COMPX</h5>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl COMPX-F Primer</li>
+                    <li>1μl COMPX-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl TOP10 Solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 55℃, extend for 1 min. 2 tubes without Taq DNA Polymerase as blank control.</p>
+                  <h5>4th Electrophoresis for Colonical PCR</h5>
+                  <p>Bands are predicted except one. Maybe caused by accident. PCR product keeped for later use.</p>
+                  <p><img src="http://chuantu.biz/t2/10/1436790740x-1376436670.jpg" alt="PCR for COMPX"></p>
+                  <h5>1st Plasmid Extraction for BBa K1492002 &amp; pSB1C3</h5>
+                  <p>1 Centifuge 5 ml bacterium solution, few sediment getted.</p>
+                  <p>2 Add 250 μl Buffer P1, resuspend cells.</p>
+                  <p>3 Add 250 μl Buffer P2, mix well, 4 min's standing.</p>
+                  <p>4 Add 350 μl Buffer P3, mix well.</p>
+                  <p>5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.</p>
+                  <p>6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.</p>
+                  <p>7 11.6 rpm centifuge 1 min.</p>
+                  <p>8 Add 50 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.</p>
+                  <h5>1st Electrophoresis for Plasmid Extraction</h5>
+                  <p>No result. May due to high concentration of chloramphenicol, bacteria grow very slow. As there is few bacteria, no plasmid getted. Bacterium solution dilute 1:1 with LB medium.</p>
+                  <h5>绿脓杆菌划线</h5>
+                  <p>大约在傍晚7点左右，利用划线法在LB平板上培养绿脓杆菌。一块是从昨天的培养的培养基里分出来的，另一块是从菌液中沾取划线的。</p>
+                  <p>10点离开前，看了一次，从培养基分出来的那一块平板已经有菌落生成了。</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-13 00:00"><span>2015</span> <span>13/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>The steak-plate of <em>Pseudomonas aeruginosa</em> </h5>
+                  <p>
+                    <br>By using the steak-plate method, we successful separated the single conlony of <em>P. aeruginosa</em>.
+                    <br>\(^o^)/~！</p>
+                  <p><img src="http://chuantu.biz/t2/10/1436798097x-1376436670.jpg" alt="PA"></p>
+                  <h5>2nd Plasmid Extraction for BBa K1492002 &amp; pSB1C3</h5>
+                  <p>1 Centifuge 4.5 ml bacterium solution.</p>
+                  <p>2 Add 250 μl Buffer P1, resuspend cells.</p>
+                  <p>3 Add 250 μl Buffer P2, mix well, 4 min's standing.</p>
+                  <p>4 Add 350 μl Buffer P3, mix well.</p>
+                  <p>5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.</p>
+                  <p>6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.</p>
+                  <p>7 11.6 rpm centifuge 1 min.</p>
+                  <p>8 Add 30 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.</p>
+                  <h5>2nd Electrophoresis for Plasmid Extraction</h5>
+                  <p>Successfully extracted tRNA plasmid. Failed to extract pSB1C3 plasmid, cause culture is contaminated.
+                    <br>Result:
+                    <br><img src="http://chuantu.biz/t2/10/1436790657x-1376436664.jpg" alt="plasmid extraction"></p>
+                  <p>tRNA 114ng/μl</p>
+                  <h5>1st PCR for tRNA</h5>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl tRNA-F2 Primer</li>
+                    <li>1μl tRNA-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl tRNA plasmid</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 50℃, extend for 1:30.</p>
+                  <h5>1st Electrophoresis for tRNA PCR</h5>
+                  <p>No result. </p>
+                  <h5>Transformation of Four Parts of Plate 4 </h5>
+                  <ol>
+                    <li>material:
+                      <br>competent cell TG1 from the lab 319
+                      <br>4 parts of the plate 4(pSB1A2)
+                      <br>No.1:BBa_B0034 No.2:BBa_C0051
+                      <br>No.3:BBa_R0051 No.4:BBa_E0040</li>
+                    <li>the procedure
+                      <br>(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
+                      <br>(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
+                      <br>(3)Put the tubes on the ice about 30 mins.
+                      <br>(4)Make a heat shock at 42 degree centigrade about 60 sec
+                      <br>(5)Put the tubes on the ice about 3 mins again.
+                      <br>(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
+                      <br>(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
+                      <br>(8)Discard the supernatant liquid and leave about 100ul medium.
+                      <br>(9)Coat plate: add 100ul solution in a plate </li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-14 00:00"><span>2015</span> <span>14/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>The Results of Transformation of Jul 13th</h5>
+                  <p>transformations of three parts are successful:
+                    <br>No.1 B0034
+                    <br> No.2 C0051
+                    <br> No.4 E0040
+                    <br>transformation of one part is failing:
+                    <br>No.3 R0051</p>
+                  <h5>Plasmid Extraction of pSB1C3</h5>
+                  <p>the plasmis:pSB1C3</p>
+                  <p>the procedure:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg.
+                    <br>PS:this protocol is from lab 319</p>
+                  <p>the concentration :11.0ng/ul
+                    <br>PS:the concentration of the control plasmid is 136.0 ng/ul,so we guess that maybe the concentration of bacterium solution is low.Also we guess that it's caused by the missing of spreading.</p>
+                  <h5>Transformation of Three Parts and One Plasmid</h5>
+                  <ol>
+                    <li>the plasmid going to be transformed:
+                      <br>No.1 BBa_R0062(plate 2;pSB1C3)
+                      <br>No.2 BBa_B0015(plate 3;pSB1C3)
+                      <br>No.3 BBa_R0051(plate 4;pSB1A2)
+                      <br>No.4 pSB1C3</li>
+                    <li>the procedure:
+                      <br>note:add 1 ul R0062 ,1 ul B0015,2 ul R0051,
+                      <br>2 ul pSB1C3 to 100 ul competent cells respectively</li>
+                  </ol>
+                  <p>(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
+                    <br>(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
+                    <br>(3)Put the tubes on the ice about 30 mins.
+                    <br>(4)Make a heat shock at 42 degree centigrade about 60 sec
+                    <br>(5)Put the tubes on the ice about 3 mins again.
+                    <br>(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
+                    <br>(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
+                    <br>(8)Discard the supernatant liquid and leave about 100ul medium.
+                    <br>(9)Coat plate: add 100ul solution in a plate </p>
+                  <h5>Plasmid Extraction of Three Parts of Plate 4</h5>
+                  <p>the procedure is the same as before
+                    <br>the concentration :
+                    <br>B0034 157.6ng/ul, 113.7ng/ul;
+                    <br>C0051 171.7ng/ul, 194.6ng/ul;
+                    <br>E0040 278.5ng/ul, 228.2ng/ul</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-15 00:00"><span>2015</span> <span>15/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Results of transformation </h5>
+                  <p>R0051 no colony
+                    <br>R0062 have single colony
+                    <br>B0015 have single colony
+                    <br>pSB1C3 have single colony and the colony is red</p>
+                  <h5>Plasmid Extraction Results</h5>
+                  <p>This is extraction was for previous transformation.</p>
+                  <ul>
+                    <li>pSB1C3(1): 108ng/uL</li>
+                    <li>pSB1C3(2): 157ng/uL</li>
+                    <li>B0015(1): 19ng/uL</li>
+                    <li>B0015(2): 93ng/uL</li>
+                  </ul>
+                  <p>Prepared for further use.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-16 00:00"><span>2015</span> <span>16/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Colonical PCR for micF and SoxS</h5>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF-luxI-R</td>
+                        <td>TTATAGTCATATAAATTTTCTCCTCTTTCCGAATGCGAGGCATCC</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F</td>
+                        <td>GCGGCCGCTTCTAGAGCCTCATTAATCAGTCGGC</td>
+                      </tr>
+                      <tr>
+                        <td>SoxS-RBS-luxI-R</td>
+                        <td>TATAGTCATATAAATTTTCTCCTCTTTCTAGTGCGTGCGCCGGTACCTT</td>
+                      </tr>
+                      <tr>
+                        <td>BB-SoxS-F</td>
+                        <td>CGGCCGCTTCTAGAGCGTGCGGAACATTCG</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>For micF, primers including micF-luxI-R, micF-F, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl micF-luxI-R Primer</li>
+                    <li>1μl micF-F Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl Top10(K-12) Strain <em>E. coli</em> bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl SoxS-RBS-luxI-R Primer</li>
+                    <li>1μl BB-SoxS-F Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl 1μl Top10(K-12) Strain <em>E. coli</em> bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Characterization of Colonical PCR by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/10/1437054155x-1376436664.jpg" alt="PCR">
+                    <br>The result illustrated the success of PCR</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-17 00:00"><span>2015</span> <span>17/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Colonical PCR for OprF from <em>Psudomonus aeruginosa</em></h5>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>OprF-F</td>
+                        <td>AGAGGAGAAAATCGGAATGAAACTGAAGAACAC</td>
+                      </tr>
+                      <tr>
+                        <td>OprF+Ter-R</td>
+                        <td>TGATGCCTGGCTCTAGTATTACTTGGCTTCAGCTT</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR protocol:</p>
+                  <p>For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl OprF-F Primer</li>
+                    <li>1μl OprF+Ter-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl <em>Psudomonus aeruginosa</em> (PAO1) bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Plasmid Extraction of SCVE</h5>
+                  <p>SCVE synthesized from Sangon Biotech Co.,Ltd.</p>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>Transformation for CRISPR</h5>
+                  <p>1.材料：competent cell TG1 from the lab 319，4Mparts of the plate 4(pSB1A2)</p>
+                  <p>2.编号：1:BBa_B0034 2:BBa_C0051</p>
+                  <p>3:BBa_R0051 4:BBa_E0040</p>
+                  <p>3.步骤：</p>
+                  <p>（1）将感受态细胞置于冰上，孵化5分钟；</p>
+                  <p>（2）取1ul 含代转parts的plasmid与感受态细胞混合，轻轻混匀；</p>
+                  <p>（3）冰上放置30min，中间轻摇数次，防止菌体沉淀；</p>
+                  <p>（4）42.0度水浴静置热激90s（这个时间控制很重要），立即放入冰上冷却2-3min；</p>
+                  <p>（5）加入900ul LB(不含抗生素)培养基，混匀；</p>
+                  <p>（6）37度振荡培养1h；</p>
+                  <p>（7）12，000rpm 离心15s，用移液器小心吸出850ul上清丢弃后，保留50ul重悬菌液;</p>
+                  <p>（8）用移液器混匀菌液，均匀涂布于含抗生素（Cl 200ml LB:50ul 100ng/ul Cl）的平板上，放入37度培养箱倒置培养过夜。</p>
+                  <h5>Characterization of SCVE and OprF by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/10/1437138201x-1376436670.jpg" alt="PCR">
+                    <br>Only 1/4 PCR results successfully duplicated results, plasimid extraction from SCVE showed well.</p>
+                  <p>Second PCR for OprF didn't work well:(</p>
+                  <h5>Digestion of E0040(EGFP)</h5>
+                  <p>Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer</p>
+                  <p>Protocol as following:</p>
+                  <ul>
+                    <li>Nuclease-free water: 15ul</li>
+                    <li>E0040: 1ul</li>
+                    <li>10* FastDigest Green Buffer: 2ul</li>
+                    <li>EcoRI: 1ul</li>
+                    <li>SpeI: 1ul</li>
+                  </ul>
+                  <p>We are trying to figure out the best conditions of our experiment and whether we need loading buffer</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Time</th>
+                        <th>With Loading Buffer or not</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>5min</td>
+                        <td>Yes(1), No(2)</td>
+                      </tr>
+                      <tr>
+                        <td>2h</td>
+                        <td>Yes(3), No(4)</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>PCR of E0040(EGFP)</h5>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>E0040-F</td>
+                        <td>ATGCGTAAAGGAGAAGAACTTT</td>
+                      </tr>
+                      <tr>
+                        <td>R0051-E0040-R</td>
+                        <td>TACGCATATAAATTTTCTCCTCTTTGCAACCA</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>For E0040, primers including E0040-F, R0051-E0040-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl E0040-F Primer</li>
+                    <li>1μl R0051-E0040-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl plasmid(pSB1C3) containing E0040</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p><em>Attention</em>: R0051-E0040-R Primer is not appropriate because of only 8 complementary base pairs.</p>
+                  <h5>Characterization of E0040 from Digestion and PCR </h5>
+                  <p><img src="http://chuantu.biz/t2/10/1437139187x-1376436670.jpg" alt="PCR">
+                    <br>Result illustrated that <em>We do need loading buffer, and 5 min do work!</em></p>
+                  <p>PCR showed more concentration of E0040!</p>
+                  <h5>Gel Extraction</h5>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>Final OD results:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Code</th>
+                        <th>OD(ng/ul)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR(1)</td>
+                        <td>15.3</td>
+                      </tr>
+                      <tr>
+                        <td>PCR(2)</td>
+                        <td>4.5</td>
+                      </tr>
+                      <tr>
+                        <td>PCR(3)</td>
+                        <td>27.0</td>
+                      </tr>
+                      <tr>
+                        <td>Digestion(1) with 5 mins</td>
+                        <td>15.4</td>
+                      </tr>
+                      <tr>
+                        <td>Digestion(3) with 2 h</td>
+                        <td>22.7</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Perseved for further use.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-18 00:00"><span>2015</span> <span>18/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Transfofmation of Three Parts</h5>
+                  <p>Protocol as following</p>
+                  <ul>
+                    <li>100μl (three tubes)competent cells</li>
+                    <li>C0062(pSB1C3)</li>
+                    <li>R0010(pSB1C3)</li>
+                    <li>C0061(pSB1C3)</li>
+                    <li>LB without antibiotic</li>
+                    <li>LB medium with Amp/CmR</li>
+                  </ul>
+                  <h5>Plasmid Extraction of E0040</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <p><em>Results</em></p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Code</th>
+                        <th>OD(ng/ul)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>E0040(1)</td>
+                        <td>166.7</td>
+                      </tr>
+                      <tr>
+                        <td>E0040(2)</td>
+                        <td>169.2</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>Colonical PCR for OprF from <em>Psudomonus aeruginosa</em></h5>
+                  <p>PCR protocol:
+                    <br>For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl OprF-F Primer</li>
+                    <li>1μl OprF+Ter-R Primer</li>
+                    <li>0.5μl Taq DNA Polymerase(<em>add more 0.25ul then previous protocol</em>)</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl <em>Psudomonus aeruginosa</em> (PAO1) bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Digestion of E0040(EGFP)</h5>
+                  <p>Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer</p>
+                  <p>Protocol as following:</p>
+                  <ul>
+                    <li>Nuclease-free water: 15ul</li>
+                    <li>E0040: 1ul</li>
+                    <li>10* FastDigest Green Buffer: 2ul</li>
+                    <li>EcoRI: 1ul</li>
+                    <li>SpeI: 1ul</li>
+                  </ul>
+                  <h5>Characterization of OprF and E0040</h5>
+                  <p><img src="http://chuantu.biz/t2/10/1437210607x-1376436670.jpg" alt="图片名称">
+                    <br>酶连效果不是很好，基本建议是改善昨日胶回收的条带进行PCR扩增，之前引物设计担心不是很好，但是看样子没有其他位点被p了出来；
+                    <br>OprF的PCR依然是在相同条件下只有一个条带p了出来，比较异常；此外引物二聚体较多，因此对此批进行了胶回收，以备后续使用。</p>
+                  <h5>Plasmid Extraction of Cas9(K1218011)</h5>
+                  <p>The bacteria were cultured at 9 am from successfully transformed bacteria. </p>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>Characterization of Cas9</h5>
+                  <p><img src="http://chuantu.biz/t2/10/1437231617x-1376436670.jpg" alt="图片名称">
+                    <br>转化成功，明天写实验结果讨论</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-19 00:00"><span>2015</span> <span>19/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Digestion of K1218011(Cas9)</h5>
+                  <p>Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer</p>
+                  <p>Protocol as following:</p>
+                  <ul>
+                    <li>Nuclease-free water: 15ul</li>
+                    <li>K1218011: 1ul</li>
+                    <li>10* FastDigest Green Buffer: 2ul</li>
+                    <li>EcoRI: 1ul</li>
+                    <li>SpeI: 1ul</li>
+                  </ul>
+                  <p>Incubate into 37 degree celcius, 2h</p>
+                  <h5>Digestion of SCVE</h5>
+                  <p>Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer</p>
+                  <p>Protocol as following:</p>
+                  <ul>
+                    <li>Nuclease-free water: 16ul</li>
+                    <li>SCVE: 1ul</li>
+                    <li>10* FastDigest Green Buffer: 2ul</li>
+                    <li>SmaI: 1ul</li>
+                  </ul>
+                  <p>Incubate into 37 degree celcius, 2h</p>
+                  <h5>Colonical PCR for OprF from *Psudomonus aeruginosa, micF and SoxS from K-12 </h5>
+                  <p>PCR protocol:</p>
+                  <p>For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl OprF-F Primer</li>
+                    <li>1μl OprF+Ter-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl <em>Psudomonus aeruginosa</em> (PAO1) bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>For micF, primers including micF-luxI-R, micF-F, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl micF-luxI-R Primer</li>
+                    <li>1μl micF-F Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl Top10(K-12) Strain <em>E. coli</em> bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl SoxS-RBS-luxI-R Primer</li>
+                    <li>1μl BB-SoxS-F Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl 1μl Top10(K-12) Strain <em>E. coli</em> bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Praparation for Competent Cell</h5>
+                  <p>protocol as the following:</p>
+                  <ol>
+                    <li>Pick out two single colonies(TG1) and cultivate bacteria in 5mL LB media at 37 degree centigrade about 12 hours with shocking. </li>
+                    <li>Take 5mL nutrient solution into 100 mL media and cultivate bacteria at 37 degree centigrade about one hour and a half with shocking till the OD660 reaches 0.4-0.5. </li>
+                    <li>Absorb 50 mL nutrient solution into EP tubes with ice-bath about 10 mins. </li>
+                    <li>Centrifuge the tubes at 4100xg at 4 degree centigrade about 10mins and discard the supernatant liquid.</li>
+                    <li>Utilize 30 mL 0.1M calcium chloride to suspend bacteria.</li>
+                    <li>Centrifuge the tubes at 4100xg at 4 degree centigrade about 10 mins and discard the supernatant liquid. </li>
+                    <li>Utilize 2 mL 0.1M calcium chloride with 15% glycerol to suspend bacteria and then ready for transformation quickly or restore at -70 degree centigrade.</li>
+                  </ol>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Time(min)</th>
+                        <th>OD600</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>40</td>
+                        <td>0.195</td>
+                      </tr>
+                      <tr>
+                        <td>60</td>
+                        <td>0.203</td>
+                      </tr>
+                      <tr>
+                        <td>80</td>
+                        <td>0.309</td>
+                      </tr>
+                      <tr>
+                        <td>90</td>
+                        <td>0.439</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PS:dilute the solution by adding 100 mL LB media after first test.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-20 00:00"><span>2015</span> <span>20/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Negative Chemotaxis Characterization</h5>
+                  <p>term 1, material:</p>
+                  <ul>
+                    <li>LB solid medium plates x4</li>
+                    <li>1g/ml Chloramphenicol</li>
+                    <li>E.coli Top10</li>
+                    <li>filter paper</li>
+                  </ul>
+                  <h5>Plasmid Extraction of EmrE and ArcB</h5>
+                  <p>done by Wang Luyao</p>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <p>the concentration as following
+                    <br>EmrE 70.2 ng/ul 92.3 ng/ul
+                    <br>ArcB 62.6 ng/ul 95.2 ng/ul</p>
+                  <h5>Transformation of R0051 and C0061</h5>
+                  <p>the source of parts:
+                    <br>No.1 R0051 2014 kit plate 4
+                    <br>No.2 R0051 2014 kit plate 4
+                    <br>No.3 control
+                    <br>No.4 C0061 2015 kit plate 4
+                    <br>No.5 C0061 2015 kit plate 4
+                    <br>No.6 C0061 2014 kit plate 4</p>
+                  <p>No.1 and No.4 the competent cell is from lab319
+                    <br>No.2 and No.5 the competent cell is made by us (firt)
+                    <br>No.3 and No.6 the competent cell is made by us (second)</p>
+                  <p>the protocol as following
+                    <br>(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
+                    <br>(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
+                    <br>(3)Put the tubes on the ice about 30 mins.
+                    <br>(4)Make a heat shock at 42 degree centigrade about 60 sec
+                    <br>(5)Put the tubes on the ice about 3 mins again.
+                    <br>(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
+                    <br>(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
+                    <br>(8)Discard the supernatant liquid and leave about 100ul medium.
+                    <br>(9)Coat plate: add 100ul solution in a plate</p>
+                  <h5>PCR of OrpF</h5>
+                  <p>material:</p>
+                  <ul>
+                    <li>13 uL Taq Master Mix </li>
+                    <li>11 uL dd water</li>
+                    <li>1 uL PAO1 bacterial</li>
+                    <li>0.5 uL primer Fd(OrpF-F)</li>
+                    <li>0.5 uL primer Rv(OrpF+Ter-R)
+                      <br>PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.</li>
+                  </ul>
+                  <p>Renaturation at 50℃, extend for 1:10</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-21 00:00"><span>2015</span> <span>21/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR of OprF</h5>
+                  <p>material:
+                    <br>No.1 and No.2:</p>
+                  <ul>
+                    <li>6.5 uL Taq Master Mix</li>
+                    <li>1 uL PAO1 bacterial solution</li>
+                    <li>4.5 uL dd water</li>
+                    <li>0.5 uL OprF-F</li>
+                    <li>0.5 uL OprF+Ter-R</li>
+                  </ul>
+                  <p>No.3 and No.4:</p>
+                  <ul>
+                    <li>6.5 uL Taq Master Mix</li>
+                    <li>4 uL OprF PCR product</li>
+                    <li>1.5 uL dd water </li>
+                    <li>0.5 uL OprF-F</li>
+                    <li>0.5 uL OprF+Ter-R</li>
+                  </ul>
+                  <p>PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.</p>
+                  <p>Renaturation at 55℃, extend for 1:10</p>
+                  <h5>OprF for PCR  and gel electrophoresisOprF</h5>
+                  <p></p>
+                  <p> <img src="http://chuantu.biz/t2/11/1437619234x-1376436670.jpg" alt="图片名称"></p>
+                  <p>analyze: the length of the product is </p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-22 00:00"><span>2015</span> <span>22/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Preparation of Semi-Solid LB Medium</h5>
+                  <p>In order to characterization the movement of TOP10, semi-solid LB medium preparation is indispensable in this research.</p>
+                  <p> (500mL Protocol)</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Mass</th>
+                        <th>Proportion(g/mL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Yeast Extract</td>
+                        <td>2.5g</td>
+                        <td>0.5%</td>
+                      </tr>
+                      <tr>
+                        <td>Trtptone</td>
+                        <td>5g</td>
+                        <td>1%</td>
+                      </tr>
+                      <tr>
+                        <td>NaCl</td>
+                        <td>5g</td>
+                        <td>1%</td>
+                      </tr>
+                      <tr>
+                        <td>Agar</td>
+                        <td>1.25g</td>
+                        <td>0.25%</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Compared with solid LB medium, containing 1.5% agar in there.</p>
+                  <h5>Plasmid Extraction C0051,B0015 and R0062</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <p>the concentration as following
+                    <br>C0015:171.7 ng/uL
+                    <br>B0015:93 ng/uL
+                    <br>R0062:144.3 ng/uL</p>
+                  <h5>PCR of C0051,B0015 and R0062</h5>
+                  <p>material:
+                    <br>C0051 plasmid(171.7 ng/uL)
+                    <br>B0015 plasmid(93 ng/uL)
+                    <br>R0062 plasmid(144.3 ng/uL)
+                    <br>all the solutions are diluted 10-folds </p>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>C0051-F(I)</td>
+                        <td>att tat atg agc aaa aag aaa cca tta tca</td>
+                      </tr>
+                      <tr>
+                        <td>C0051-B0015-R(I)</td>
+                        <td>tat ttg atg cct ggc tct agt gat cta cac tag cac ta</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R(I)</td>
+                        <td>gcc gct act agt taa acg cag aaa ggc cca cc</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-F</td>
+                        <td>cca ggc atc aaa taa aac gaa agg</td>
+                      </tr>
+                      <tr>
+                        <td>R0062-F</td>
+                        <td>gcg gcc gct tct aga gac ctg tag gat cg</td>
+                      </tr>
+                      <tr>
+                        <td>R0062-C0051-R</td>
+                        <td>tgt gct cat ata aat ttt ctc ctc ttt cta gat ttt att cga c</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>No.1 and No.2 for C0051(803bp)
+                    <br>Protocol as this:</p>
+                  <ul>
+                    <li>2.5 μl Taq buffer with KCl</li>
+                    <li>2.5 μl dNTPs</li>
+                    <li>0.75 μl C0051-F Primer</li>
+                    <li>0.75 μl C0051-B0015-R(I) Primer</li>
+                    <li>0.5 μl Taq DNA Polymerase</li>
+                    <li>1.0 μl MgCl2</li>
+                    <li>2 uL C0051 solution</li>
+                    <li>15 μl ddH2O</li>
+                  </ul>
+                  <p>No.3 and No.4 for B0015(142bp)
+                    <br>Protocol as this:</p>
+                  <ul>
+                    <li>2.5 μl Taq buffer with KCl</li>
+                    <li>2.5 μl dNTPs</li>
+                    <li>0.75 μl B0015-F Primer</li>
+                    <li>0.75 μl B0015-R(I) Primer</li>
+                    <li>0.5 μl Taq DNA Polymerase</li>
+                    <li>1.0 μl MgCl2</li>
+                    <li>3 uL C0051 solution</li>
+                    <li>14 μl ddH2O</li>
+                  </ul>
+                  <p>No.5 and No.6 for R0062(104bp)
+                    <br>Protocol as this:</p>
+                  <ul>
+                    <li>2.5 μl Taq buffer with KCl</li>
+                    <li>2.5 μl dNTPs</li>
+                    <li>0.75 μl R0062-F Primer</li>
+                    <li>0.75 μl R0062-C0051-R Primer</li>
+                    <li>0.5 μl Taq DNA Polymerase</li>
+                    <li>1.0 μl MgCl2</li>
+                    <li>2 uL R0062 solution</li>
+                    <li>15 μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Preparation Semi-Solid M63 Medium</h5>
+                  <p>In order to characterization the movement of TOP10, semi-solid M63 medium preparation is indispensable in this research.</p>
+                  <p> (500mL Protocol) </p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Mass</th>
+                        <th>Proportion(g/mL) or Mole</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Yeast Extract</td>
+                        <td>2.5g</td>
+                        <td>0.5%</td>
+                      </tr>
+                      <tr>
+                        <td>Trtptone</td>
+                        <td>5g</td>
+                        <td>1%</td>
+                      </tr>
+                      <tr>
+                        <td>NaCl</td>
+                        <td>5g</td>
+                        <td>1%</td>
+                      </tr>
+                      <tr>
+                        <td>Agar</td>
+                        <td>1.25g</td>
+                        <td>0.25%</td>
+                      </tr>
+                      <tr>
+                        <td>KH2PO4</td>
+                        <td>6.8g</td>
+                        <td>50mM</td>
+                      </tr>
+                      <tr>
+                        <td>KOH</td>
+                        <td>2.1g</td>
+                        <td>37.5mM</td>
+                      </tr>
+                      <tr>
+                        <td>(NH4)2SO4</td>
+                        <td>0.09g</td>
+                        <td>7.5mM</td>
+                      </tr>
+                      <tr>
+                        <td>MgSO4</td>
+                        <td>0.06g</td>
+                        <td>0.5mM</td>
+                      </tr>
+                      <tr>
+                        <td>FeSO4·7H2O</td>
+                        <td>0.2710g</td>
+                        <td>1.95mM</td>
+                      </tr>
+                      <tr>
+                        <td>D-Glucose</td>
+                        <td>1.98g</td>
+                        <td>11mM</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>Colonical PCR for OprF from <em>Psudomonus aeruginosa</em> and SCVE from pUC57</h5>
+                  <p>PCR protocol:</p>
+                  <p>For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl OprF-F Primer</li>
+                    <li>1μl OprF+Ter-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl <em>Psudomonus aeruginosa</em> (PAO1) bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>For SCVE, Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>SCVE-T-R</td>
+                        <td>TTGATGCCTGGCTCTAGTACACCAGCAGATCCGG</td>
+                      </tr>
+                      <tr>
+                        <td>T7-RBS-SCVE-F</td>
+                        <td>TTTTTGCATACTAGAGAAAGAGGAGAAAATTTATATGTATAGCTTTGTG</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Protocol as this:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl SCVE-T-R Primer</li>
+                    <li>1μl T7-RBS-SCVE-F Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl SCVE solution from plasmid extraction</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Electrophoresis of OprF, SCVE micF and SoxS</h5>
+                  <p>micF and SoxS
+                    <br><img src="http://chuantu.biz/t2/11/1437619293x-1376436670.jpg" alt="图片名称"></p>
+                  <p>OprF and SCVE
+                    <br><img src="http://chuantu.biz/t2/11/1437619380x-1376436670.jpg" alt="图片名称"></p>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: OprF, SoxS, micF</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-23 00:00"><span>2015</span> <span>23/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Plasmid Extraction of R0062</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>PCR for OprF and C0061</h5>
+                  <p>PCR protocol:</p>
+                  <p>For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl OprF-F Primer</li>
+                    <li>1μl OprF+Ter-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl <em>Psudomonus aeruginosa</em> (PAO1) bacterial solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>For SCVE, Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>C0061-F</td>
+                        <td>ATGACTATAATGATAAAAAAATCGGATTTTTTGGCA</td>
+                      </tr>
+                      <tr>
+                        <td>C0061-B0015-R</td>
+                        <td>ATTTGATGCCTGGGAGATCTACACTAGC</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Protocol as this:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl C0061-F Primer</li>
+                    <li>1μl C0061-B0015-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1μl C0061 solution</li>
+                    <li>12.35μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Extraction of C0062,B0015 and R0062</h5>
+                  <p>Material including: C0051,B0015 and R0062</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>the concentration as following:
+                    <br>C0051:12.7 ng/uL
+                    <br>B0015:7.3 ng/uL
+                    <br>R0062:7.7 ng/uL</p>
+                  <h5>PCR of OprF</h5>
+                  <p>material:</p>
+                  <ul>
+                    <li>13 uL Taq Master Mix </li>
+                    <li>11 uL dd water</li>
+                    <li>1 uL PAO1 bacterial</li>
+                    <li>0.5 uL primer Fd(OrpF-F)</li>
+                    <li>0.5 uL primer Rv(OrpF+Ter-R)</li>
+                  </ul>
+                  <p>Renaturation at 55℃, extend for 2min</p>
+                  <h5>Gel Electrophoresis Characterization</h5>
+                  <p>C0051 and B0015
+                    <br><img src="http://chuantu.biz/t2/11/1437640573x1822614202.jpg" alt="图片名称"></p>
+                  <p>OprF
+                    <br><img src="http://chuantu.biz/t2/11/1437640593x1822614202.jpg" alt="图片名称">
+                    <br>That was relatively successully extracted OprF from colonical PCR.</p>
+                  <p>R0062
+                    <br><img src="http://chuantu.biz/t2/11/1437640611x1822614202.jpg" alt="图片名称"></p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-24 00:00"><span>2015</span> <span>24/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Preparation for Homologous Recombination: Plasimid Digestion</h5>
+                  <p></p>
+                  <p>Enzyme from <em>Thermo Scientific FastDigest</em> Enzyme with 10X Green Buffer</p>
+                  <p>Protocol as following:</p>
+                  <ul>
+                    <li>Nuclease-free water: 15ul</li>
+                    <li>pSB1C3: 1ul</li>
+                    <li>10* FastDigest Green Buffer: 2ul</li>
+                    <li>EcoRI: 1ul</li>
+                    <li>SpeI: 1ul</li>
+                  </ul>
+                  <p>Incubate solution at 37 degree celcius about 2h.</p>
+                  <h5>PCR of C0061,B0015 and micF</h5>
+                  <p>material:
+                    <br>C0061 plasmid: 477.1 ng/uL(diluted 20-folds)
+                    <br>B0015 PCR product:7.3 ng/uL
+                    <br>micF-1 PCR product:13.4 ng/uL</p>
+                  <p>ForB0015 Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>B0015-F</td>
+                        <td>cca ggc atc aaa taa aac gaa agg</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R(I)</td>
+                        <td>gcc gct act agt ata taa acg cag aaa ggc cca cc</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>protocol:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl Primer-Fd</li>
+                    <li>1μl Primer-Rv</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>1uL C0061 plasmid,4uL B0015 plasmid,2uL plasmid</li>
+                    <li>(13.35-X)μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 55℃, extend for 1min</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-25 00:00"><span>2015</span> <span>25/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR of C0061,B0015,R0062,R0010 and C0062 </h5>
+                  <p>material:
+                    <br>C0061 plasmid:477.1 ng/uL(0.2 uL)
+                    <br>B0015 plasmid:19 ng/uL(3 uL)
+                    <br>R0062 plasmid:143.3 ng/uL(0.5 uL)
+                    <br>C0051 plasmid:171.7 ng/uL(0.5 uL)
+                    <br>R0010 plasmid:357.7 ng/uL(diluted 10-folds,1 uL)
+                    <br>C0062 plasmid:739.4 ng/uL(diluted 10-folds,1uL)
+                    <br> for R0062.R0010 and C0062Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>R0062-F</td>
+                        <td>gcg gcc gct tct aga ctg tag gat cg</td>
+                      </tr>
+                      <tr>
+                        <td>R0062-C0051-R</td>
+                        <td>tgt gct cat ata aat ttt ctc ttt cta gat ttt att cga c</td>
+                      </tr>
+                      <tr>
+                        <td>backbone-R0010-F</td>
+                        <td>ccgctt cta gag cca tac gca aac cgc ctc tc</td>
+                      </tr>
+                      <tr>
+                        <td>C0062-B0034-R</td>
+                        <td>cta gtg tc agt aat aaa ttt tct cct ctt tg tgt gaa att gt</td>
+                      </tr>
+                      <tr>
+                        <td>B0034-C0062-F</td>
+                        <td>tat tac tga gca cta gat gaa aaa cat aaa tgc cga c</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-C0062-R</td>
+                        <td>atg cct vggc tct agt gat cta cac tag cac t</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>protocol:</p>
+                  <ul>
+                    <li>2μl Taq buffer with KCl</li>
+                    <li>0.4μl dNTPs</li>
+                    <li>1μl Primer-Fd</li>
+                    <li>1μl Primer-Rv</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>2μl MgCl2</li>
+                    <li>XuL plasmid</li>
+                    <li>(13.35-X)μl ddH2O</li>
+                  </ul>
+                  <p>Renaturation at 55℃, extend for 1min
+                    <br>PS:
+                    <br>C0061:656bp B0015:142bp R0062:104bp
+                    <br>C0051:803bp C0062:813bp R0010:243bp</p>
+                  <h5>Characterization of micF and SoxS by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/11/1437914340x-1566701588.jpg" alt="图片名称">
+                    <br>micF were successfully PCRed.</p>
+                  <h5>Characterization of pSB1C3 and SoxS by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/11/1437914405x-1566701588.jpg" alt="图片名称"></p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-26 00:00"><span>2015</span> <span>26/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Characterization of R0051, C0061 and C0051 by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/11/1437914472x-1566701588.jpg" alt="图片名称"></p>
+                  <h5>Plasimid Extraction for pSB1C3 and C0061</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: R0051, C0051 and C0061</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <h5>Characterization of B0015 and R0051 by Gel Electrophoresis</h5>
+                  <p><img src="http://chuantu.biz/t2/11/1437914553x-1566701588.jpg" alt="图片名称"></p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-27 00:00"><span>2015</span> <span>27/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Overlap Circuit</h5>
+                  <p><img src="http://chuantu.biz/t2/10/1436688293x-1376436670.png" alt="图片名称">
+                    <br>This sensing circuit including promoter micF and SoxS.</p>
+                  <h5>Overlapping Fragments</h5>
+                  <p>In order to ligate the fragements micF or SoxS, C0061 and B0015, we need to make the equal mole proportion.</p>
+                  <p>The mole of DNA molecule is calculated in the following ways:</p>
+                  <p><img src="http://chuantu.biz/t2/11/1437963054x-1566638177.gif" alt="图片名称"></p>
+                  <p>among which, c is the concentration of DNA, V is the volumn required and n is the length of DNA. Because of the same molar mass of As, Gs, Cs and Ts, computation of same molar mass is omitted.</p>
+                  <p>According to our sample extracted from PCR and calculation, ingradients characterize as below:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Concentration(ng/uL)</th>
+                        <th>Length(bp)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF</td>
+                        <td>12.6</td>
+                        <td>358</td>
+                      </tr>
+                      <tr>
+                        <td>SoxS</td>
+                        <td>27.5</td>
+                        <td>705</td>
+                      </tr>
+                      <tr>
+                        <td>C0061(LuxI)</td>
+                        <td>18.5</td>
+                        <td>656</td>
+                      </tr>
+                      <tr>
+                        <td>B0015(Terminator)</td>
+                        <td>9.5</td>
+                        <td>143</td>
+                      </tr>
+                      <tr>
+                        <td>Total</td>
+                        <td></td>
+                        <td>1161</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>The theoratical volumn proportion is: </p>
+                  <ul>
+                    <li>SoxS-C0061-B0015: 5: 7: 3</li>
+                    <li>micF-C0061-B0015: 5.5: 7: 3</li>
+                  </ul>
+                  <p>Firstly, to ligate these parts, we need 10 cycles without primer:
+                    <br>for circuit SoxS-C0061-B0015:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>SoxS</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>7</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>3</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>0.35</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for circuit micF-C0061-B0015:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF</td>
+                        <td>5.5</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>7</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>3</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Then, we extracted 5 ul as template, and 15 ul adding with primer:
+                    <br>Primers were synthesized from <em>Sangon Biotech. Co.,Ltd.</em></p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF-F</td>
+                        <td>GCGGCCGCTTCTAGAGCCTCATTAATCAGTCGGC</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R</td>
+                        <td>GCCGCTACTAGTATATAAACGCAG</td>
+                      </tr>
+                      <tr>
+                        <td>BB-SoxS-F</td>
+                        <td>CGGCCGCTTCTAGAGCGTGCGGAACATTCG</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>5ul-template protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Template</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F(BB-SoxS-F)</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>8.35</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>15ul-protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Solution</td>
+                        <td>15</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F(BB-SoxS-F)</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>1</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of Five Parts</h5>
+                  <p>material:
+                    <br>R0010 plasmid:193.1ng/uL
+                    <br>C0062 plasmid:952.5ng/uL
+                    <br>B0015 plasmid:93ng/uL
+                    <br>R0062 plasmid：143.3ng/uL</p>
+                  <p>For R0051 Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <p>protocol:</p>
+                  <ul>
+                    <li>2uL 10X buffer with KCl</li>
+                    <li>0.4uL dNTP</li>
+                    <li>1uL primers-F</li>
+                    <li>1uL primers-R</li>
+                    <li>0.25uL Taq Polymerase</li>
+                    <li>2uL MgCl2</li>
+                    <li>1uL plasmid</li>
+                    <li>12.35 uL ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Overlap PCR of bacteria III</h5>
+                  <p>material:</p>
+                  <ul>
+                    <li>R0062 PCR product:21.2 ng/uL</li>
+                    <li>C0051 PCR product:22.8 ng/uL</li>
+                    <li>B0015 PCR product:24.6 ng/uL</li>
+                  </ul>
+                  <p>protocol:
+                    <br>total volume 20 uL </p>
+                  <ul>
+                    <li>R0062(104bp) 1 uL</li>
+                    <li>C0051(803bp) 8 uL</li>
+                    <li>B0015(153bp) 1.5uL</li>
+                    <li>ddH2O 4.85 uL</li>
+                    <li>buffer 2uL</li>
+                    <li>dNTP 0.4uL</li>
+                    <li>Taq polymerase 0.25uL</li>
+                    <li>MgCl2 2uL</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
+                    <br>(R0062-F primer 1uL;C0062-B0015-R(I) primer 1uL)to the remaining 15 uL PCR product.</p>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Result:
+                    <br> we don't get the product we want.We guess the templates are not the right products,maybe they have mutations .So we decide to do the overlap PCR of bateriaI to test whether the method is suitable.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-28 00:00"><span>2015</span> <span>28/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Plasmid Extraction of gRNA-AcrB and gRNA-EmrE</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: R0061</p>
+                  <h5><em>Case 1: Sangon Biotech.</em></h5>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <h5><em>Case 2: Axygen</em></h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add 400~600 ul Buffer DE-A and then incubate the EP tubes in thermostat water bath about 75 degree centigrade within 10 mins. </li>
+                    <li>After gel is all dissolved, add 1/3 volumn of Buffer DE-B into EP tubes. The solution will turn yellow, illustrating complete dissolved.</li>
+                    <li>Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul Buffer W1 in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Add 700 ul Buffer W2 in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <h5>Overlapping Fragments</h5>
+                  <p>We conduct the overlapping again.
+                    <br>However, the sample has a little difference. The concentration of micF changes to 58.7ng/uL. According to our sample extracted from PCR and calculation, ingradients characterize as below:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Concentration(ng/uL)</th>
+                        <th>Length(bp)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF</td>
+                        <td>57.8</td>
+                        <td>358</td>
+                      </tr>
+                      <tr>
+                        <td>SoxS</td>
+                        <td>27.5</td>
+                        <td>705</td>
+                      </tr>
+                      <tr>
+                        <td>C0061(LuxI)</td>
+                        <td>18.5</td>
+                        <td>656</td>
+                      </tr>
+                      <tr>
+                        <td>B0015(Terminator)</td>
+                        <td>9.5</td>
+                        <td>143</td>
+                      </tr>
+                      <tr>
+                        <td>Total</td>
+                        <td></td>
+                        <td>1161</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>The theoratical volumn proportion is: </p>
+                  <ul>
+                    <li>SoxS-C0061-B0015: 5: 7: 3</li>
+                    <li>micF-C0061-B0015: 1.3: 7: 3</li>
+                  </ul>
+                  <p>Firstly, to ligate these parts, we need 10 cycles without primer:
+                    <br>for circuit SoxS-C0061-B0015:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>SoxS</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>7</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>3</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>0.35</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for circuit micF-C0061-B0015:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>micF</td>
+                        <td>1.3</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>7</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>3</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>4.1</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Primers are the same as before.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Template</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP</td>
+                        <td>0.4</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F(BB-SoxS-F)</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>8.35</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>15ul-protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Solution</td>
+                        <td>15</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F(BB-SoxS-F)</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>B0015-R</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer with KCl</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>1</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of Cas9(80l)</h5>
+                  <p>material:</p>
+                  <p>8 uL buffer with KCl
+                    <br>35 uL dd water
+                    <br>8 uL Plasmid with Cas9
+                    <br>4 uL primer R
+                    <br>4 uL primer F
+                    <br>8 uL MgCl2
+                    <br>8 uL dNTPs
+                    <br>5 uL DMSO
+                    <br>1 uL Tap polymerase</p>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p><img src="http://chuantu.biz/t2/11/1438139367x-954497725.jpg" alt="图片名称"></p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-29 00:00"><span>2015</span> <span>29/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Negative Chemotaxis Characterization</h5>
+                  <p>Done by Wang Luyao
+                    <br><em>Term 1 results</em></p>
+                  <p><img src="https://attachments.tower.im/tower/4c0f34dc48d04526869c32e9647ffa0e?version=auto&amp;filename=IMG_3119.JPG" alt="IMG_3119.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/5d6b4f62cd8b42feadcc7c876941da67?version=auto&amp;filename=IMG_3117.JPG" alt="IMG_3117.JPG"></p>
+                  <p> I put the paper on this plate after the E.coli grows homogeneously， so let's see the result later.</p>
+                  <p><img src="https://attachments.tower.im/tower/78199a41410b47b6a4a63fdb74822a06?version=auto&amp;filename=IMG_3121.JPG" alt="IMG_3121.JPG"></p>
+                  <p>the controlled plate</p>
+                  <p>the last plate was contaminated</p>
+                  <p><em>Term 2</em></p>
+                  <p>material:</p>
+                  <p>LB solid medium plates x6</p>
+                  <p>0.8、0.6、0.4、0.2、0.1g/ml Chloramphenicol</p>
+                  <p>E.coli Top10</p>
+                  <p>filter paper</p>
+                  <h5>Enzyme Digestion</h5>
+                  <p>Performing twice separately</p>
+                  <p>Both as following protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>pSB1C3</td>
+                        <td>10</td>
+                      </tr>
+                      <tr>
+                        <td>XbaI</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>SpeI</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>green buffer</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>6</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>Overlap PCR of bacteriaI</h5>
+                  <p>material:</p>
+                  <ul>
+                    <li>micF PCR product :33.6ng/uL</li>
+                    <li>C0061 PCR product: 18.5ng/uL</li>
+                    <li>B0015 PCR product:9.2ng/uL</li>
+                    <li>micF-F primer</li>
+                    <li>B0015-R primer</li>
+                  </ul>
+                  <p>protocol:
+                    <br>total volume: 25uL</p>
+                  <ul>
+                    <li>Taq Master Mix 12.5uL</li>
+                    <li>micF 2uL</li>
+                    <li>C0061 6uL</li>
+                    <li>B0015 2.6uL</li>
+                    <li>ddH2O 1.9uL</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
+                    <br>to the remaining 15 uL PCR product.</p>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Results:
+                    <br>We don't get the product we want.We make some research and guess that the probable reasons are the following:Taq polymerase can cause 3'd A to PCR product;the annealing temperature is unsuitable;the length of overlap fragment isn't enough .</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-30 00:00"><span>2015</span> <span>30/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Negative Chemotaxis Characterization</h5>
+                  <p>Done by Wang Luyao
+                    <br>7.30</p>
+                  <p><em>Term 2 results</em></p>
+                  <p><img src="https://attachments.tower.im/tower/1929f0f1a6af4fcb8c7cf254920c8f91?version=auto&amp;filename=ee%20001.JPG" alt="ee 001.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/7fdbad45955c4d72b3327362eab10552?version=auto&amp;filename=qqwqw%20003.JPG" alt="qqwqw 003.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/3957a621d73b4dd28532dea571f157dd?version=auto&amp;filename=ee%20002.JPG" alt="ee 002.JPG"></p>
+                  <p>The 0.8、0.6、0.4 plates just look similar, the denser Chloramphenicol the paper hold， the bigger the clear zone is.
+                    <br><img src="https://attachments.tower.im/tower/2c1b10f8c093490f8d3036cbb803ddfc?version=auto&amp;filename=IMG_3125.JPG" alt="https://attachments.tower.im/tower/2c1b10f8c093490f8d3036cbb803ddfc?version=auto&amp;filename=IMG_3125.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/b4952ac7e2754892b9a40cdef1949311?version=auto&amp;filename=IMG_3126.JPG" alt="IMG_3126.JPG"></p>
+                  <p>but the 0.2 and 0.1g/ml plates are different, Top10 grows denser on the edge of the clear zone, I supposed it can show that E.coli has moved. So next time I'll try under 0.2g/ml.
+                    <br><img src="https://attachments.tower.im/tower/ea32b2fe366c4426b07ab25fe3781907?version=auto&amp;filename=IMG_3128.JPG" alt="IMG_3128.JPG"></p>
+                  <p>the controlled plate</p>
+                    <h5>PCR of C0062 and R0010</h5>
+                    <p>material:</p>
+                  <ul>
+                    <li>10 ul Taq PCR Master Mix</li>
+                    <li>7 ul ddH2O</li>
+                    <li>1 ul Primer F</li>
+                    <li>1 ul Primer R</li>
+                    <li>1 ul C0062 or R0010</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-07-31 00:00"><span>2015</span> <span>31/7</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Negative Chemotaxis Characterization</h5>
+                  <p>Done by Wang Luyao
+                    <br><em>Term 3</em>, material:</p>
+                  <ul>
+                    <li>
+                      <p>LB solid medium plates x5</p>
+                    </li>
+                    <li>
+                      <p>0.2、0.1、0.05、0.025g/ml Chloramphenicol</p>
+                    </li>
+                    <li>
+                      <p>E.coli Top10</p>
+                    </li>
+                    <li>
+                      <p>filter paper</p>
+                    </li>
+                  </ul>
+                  <h5>PCR of micF, SoxS, C0061 and R0015</h5>
+                  <p>material(volume 50uL):</p>
+                  <ul>
+                    <li>25 ul Taq PCR Master Mix</li>
+                    <li>15 ul ddH2O</li>
+                    <li>2.5 ul Primer F</li>
+                    <li>2.5 ul Primer R</li>
+                    <li>2.5 ul Top10 culture, Top10 culture, C0061 or R0015</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of tRNA</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>tRNA-F</td>
+                        <td>GAATTCGCGGCCGCTTCTAGATAATACGACTCACTATAGGGAATACAAGCTACTTGTTCTTTTTGCAATGGACGAGTTCGAAATGATT</td>
+                      </tr>
+                      <tr>
+                        <td>tRNA-R</td>
+                        <td>CTGCAGCGGCCGCTACTAGTTTACAGACGTTTGCGAATTG</td>
+                      </tr>
+                      <tr>
+                        <td>tRNA-F2</td>
+                        <td>TTTAATCCCCGTCCACTGGGTAATACGACTCACTATAGGGAATAC</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Protocol (volume 20uL):</p>
+                  <ul>
+                    <li>2 ul Taq Buffer</li>
+                    <li>0.4 ul dNTP</li>
+                    <li>1 ul tRNA-F</li>
+                    <li>1 ul tRNA-R</li>
+                    <li>0.25 ul Taq DNA Polymerase</li>
+                    <li>2 ul MgCl2</li>
+                    <li>1 ul tRNA</li>
+                    <li>12.35 ul ddH2O</li>
+                  </ul>
+                  <p>Protocol (volume 20uL):</p>
+                  <ul>
+                    <li>2 ul Taq Buffer</li>
+                    <li>0.4 ul dNTP</li>
+                    <li>1 ul tRNA-F2</li>
+                    <li>1 ul tRNA-R</li>
+                    <li>0.25 ul Taq DNA Polymerase</li>
+                    <li>2 ul MgCl2</li>
+                    <li>1 ul tRNA</li>
+                    <li>12.35 ul ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>25 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Result
+                    <br>Only one sample get PCR product, concentration 441ng/μL. tRNA-F2 dosen't work for wrong template.
+                    <br>Remember to review gene map before experiments!</p>
+                  <h5>PCR of cheZ</h5>
+                  <p>material:
+                    <br>Top 10 Bacterial Solution</p>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>GTGCAATTGATCCAGGAGCTCAGCCAGGC</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-T7-R</td>
+                        <td>GCTCCTGGATCAATTGCACATAAATTTTCTCCTCTTTTGCAAAAAGAA</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for cheZ (~600bp)
+                    <br>Protocol as this:</p>
+                  <ul>
+                    <li>2.5 μl Taq buffer with KCl</li>
+                    <li>2.5 μl dNTPs</li>
+                    <li>0.75 μl cheZ-F Primer</li>
+                    <li>0.75 μl cheZ-T7-R Primer</li>
+                    <li>0.5 μl Kod DNA Polymerase(600bp/min)</li>
+                    <li>1.0 μl MgCl2</li>
+                    <li>2 uL Top10 Solution</li>
+                    <li>15 μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min 30s)</li>
+                    <li>Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>It turned out we used wrong primer:(</p>
+                  <h5>PCR of CheZ and B0015</h5>
+                  <p>material:</p>
+                  <ul>
+                    <li>TOP 10 baterial solution</li>
+                    <li>PAO1 bacterial solution </li>
+                    <li>B0015 plasmid :93.0 ng/uL</li>
+                  </ul>
+                  <p>protocol:
+                    <br>total system 25uL</p>
+                  <ul>
+                    <li>buffer 2.5 uL</li>
+                    <li>dNTPs 2.5uL</li>
+                    <li>MgSO4 2uL</li>
+                    <li>primer-F 0.75 uL</li>
+                    <li>primer-R 0.75 uL</li>
+                    <li>bacterial solution/plasmid 1uL</li>
+                    <li>KOD-Plus enzyme 0.5uL</li>
+                    <li>ddH2O 17uL</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 2min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 15s; annealing(55 degree celcius, 30s and elongation(68 degree celcius 50s)</li>
+                    <li>Elongation for whole at 68 degree celcius 7min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>The protocol is from lab319,the enzyme is KOD-Plus.</p>
+                  <p>Result:
+                    <br> We don't get the product we want .It turned out because of wrong primer we implemented.</p>
+                  <h5>Transformation of C0062</h5>
+                  <p>the protocol as following
+                    <br>(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
+                    <br>(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
+                    <br>(3)Put the tubes on the ice about 30 mins.
+                    <br>(4)Make a heat shock at 42 degree centigrade about 60 sec
+                    <br>(5)Put the tubes on the ice about 3 mins again.
+                    <br>(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
+                    <br>(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
+                    <br>(8)Discard the supernatant liquid and leave about 100ul medium.
+                    <br>(9)Coat plate: add 100ul solution in a plate</p>
+                </div>
+              </li>
+          	</ul>
+          </div>
+        </div>
+      </div>
+    </div>
-<p> Document the dates you worked on your project.</p>
+    <div id="Daily-Notes-Stage-2" class="row">
+      <div class="card hoverable">
+        <div class="col s12 m9">
+        	<div class="card-content">
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-01 00:00"><span>2015</span> <span>1/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Negative Chemotaxis Characterzation</h5>
+                  <p>Done by Wang Luyao
+                    <br><em>Term 3 results</em></p>
+                  <p><img src="https://attachments.tower.im/tower/90a1fb3c242b4a769df1f5f7bbf1252b?version=auto&amp;filename=qqqq%20031.JPG" alt="qqqq 031.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/9a9cc17e6a794e058b55fb818f2a8dc9?version=auto&amp;filename=qqqq%20032.JPG" alt="qqqq 032.JPG"></p>
+                  <p><img src="https://attachments.tower.im/tower/b3dd3196982349b89e7b0bfac8075581?version=auto&amp;filename=qqqq%20033.JPG" alt="qqqq 033.JPG">
+                    <br><img src="https://attachments.tower.im/tower/e0e1a878c6714ba8bf0784472f61e340?version=auto&amp;filename=qqqq%20034.JPG" alt="qqqq 034.JPG"></p>
+                  <p>this time I got the similar results, but I cannot tell whether the bacteria is moved or didn't grow up.</p>
+                  <p>so let's see the term 1 result:</p>
+                  <p><img src="https://attachments.tower.im/tower/24fb626f6e294a918bb2f1a8bc09c371?version=auto&amp;filename=qqwqw%20002.JPG" alt="qqwqw 002.JPG"></p>
+                  <p> I put the paper on this plate after the E.coli grows homogeneously. Now after I took it out I got this.</p>
+                  <p>It's obviously that the bacteria moved. If E.coli died, the medium cannot be clear, the bodies will stay. In the middle of the area, there was a bubble formed by the medium and the wet paper. There is dilute Chloramphenicol, so the some bacteria moved there.</p>
+                  <p>So we qualitatively proved that E.coli negative chemotaxis !!!</p>
+                  <h5>PCR of cheZ</h5>
+                  <p>Material:
+                    <br>Top 10 Bacterial Solution</p>
+                  <p>Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>GTGCAATTGATCCAGGAGCTCAGCCAGGC</td>
+                      </tr>
+                      <tr>
+                        <td>che-Ter-R</td>
+                        <td>TTTATTTGATGCCTGGCTCTAGTATCAGAAACCCAGGCTGGACA</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for cheZ (~600bp)
+                    <br>Protocol as this:</p>
+                  <ul>
+                    <li>5 μl Taq buffer with KCl</li>
+                    <li>1 μl dNTPs</li>
+                    <li>2.5 μl cheZ-F Primer</li>
+                    <li>2.5 μl che-Ter-R Primer</li>
+                    <li>0.65 μl Pfu DNA Polymerase(600bp/min)</li>
+                    <li>5 μl MgCl2</li>
+                    <li>2.5 uL Top10 Solution</li>
+                    <li>31 μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min 30s)
+                      <br>1.Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p><em>No results: May be because of Pfu enzyme has already been out of date.</em></p>
+                  <p>Protocol again:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>2X Taq Master Mix</td>
+                        <td>25</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>che-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Top10 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>15</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>2X Taq Master Mix</td>
+                        <td>25</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>che-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Top10 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>12.5</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>2.5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p><em>No results：it turns out cheZ sequence in TOP10 is different from PAO1, we designed after PAO1</em></p>
+                  <p>Protocol again:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>2X Taq Master Mix</td>
+                        <td>25</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>che-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>15</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Taq buffer with KCl</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>che-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>28.35</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.65</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of tRNA</h5>
+                  <p>Protocol (volume 20uL):</p>
+                  <ul>
+                    <li>2 ul Taq Buffer</li>
+                    <li>0.4 ul dNTP</li>
+                    <li>1 ul tRNA-F2</li>
+                    <li>1 ul tRNA-R</li>
+                    <li>0.25 ul Taq DNA Polymerase</li>
+                    <li>2 ul MgCl2</li>
+                    <li>1 ul tRNA</li>
+                    <li>12.35 ul ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Extraction of tRNA</h5>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Add 1.5mL Buffer B2 and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 20uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 10 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>Result
+                    <br>Concentration 64ng/ul.</p>
+                  <h5>PCR of CheZ</h5>
+                  <p>material:
+                    <br>Top 10 Bacterial Solution</p>
+                  <p>protocol:
+                    <br>total system 25uL</p>
+                  <ul>
+                    <li>buffer 2.5 uL</li>
+                    <li>dNTPs 2.5uL</li>
+                    <li>MgSO4 3uL</li>
+                    <li>primer-F 0.75 uL</li>
+                    <li>primer-R 0.75 uL</li>
+                    <li>bacterial solution/plasmid 1uL</li>
+                    <li>KOD-Plus-Neo enzyme 0.5uL</li>
+                    <li>ddH2O 16uL</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 2min</li>
+                    <li>35 cycles containing: degradation(98 degree celcius, 10s; annealing(55 degree celcius, 30s and elongation(68 degree celcius 30s)</li>
+                    <li>Elongation for whole at 68 degree celcius 7min.</li>
+                    <li>4 celcius degree for preserveation.
+                      <br>The protocol is from lab 319.</li>
+                  </ol>
+                  <h5>PCR of T7 promoter</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>T7-F</td>
+                        <td>GAATTCGCGGCCGCTTCTAG</td>
+                      </tr>
+                      <tr>
+                        <td>T7-R2</td>
+                        <td>agacatgcaatttttttcattccgattttctcctcttttgcaaaaagaacaagtagct</td>
+                      </tr>
+                      <tr>
+                        <td>T7 Promoter1</td>
+                        <td>GAATTCGCGGCCGCTTCTAGAtaatacgactcactatagggaatacaagctacttgttctttttgca</td>
+                      </tr>
+                      <tr>
+                        <td>T7 Promoter2</td>
+                        <td>tgcaaaaagaacaagtagcttgtattccctatagtgagtcgtattaTCTAGAAGCGGCCGCGAATTC</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Protocol (volume 20uL):</p>
+                  <ul>
+                    <li>10 ul Mix</li>
+                    <li>1 ul T7-F</li>
+                    <li>1 ul T7-R2</li>
+                    <li>1 ul T7 Promoter1</li>
+                    <li>1 ul T7 promoter2</li>
+                    <li>6 ul ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 30s)</li>
+                    <li>Elongation for whole at 72 degree celcius 7min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR Purification of T7</h5>
+                  <ol>
+                    <li>Add Buffer B3 in 5 times volumn of PCR product.</li>
+                    <li>Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>Result
+                    <br>Concentration 28ng/ul.</p>
+                  <h5>PCR of T7 promoter</h5>
+                  <p>Conduct the PCR again.</p>
+                  <p>Protocol (volume 20uL):</p>
+                  <ul>
+                    <li>10 ul Mix</li>
+                    <li>1 ul T7-F</li>
+                    <li>1 ul T7-R2</li>
+                    <li>1 ul T7 Promoter1</li>
+                    <li>1 ul T7 promoter2</li>
+                    <li>6 ul ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 30s)</li>
+                    <li>Elongation for whole at 72 degree celcius 7min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Seamless Clone of tRNA Plasmid</h5>
+                  <p>Protocol:</p>
+                  <ul>
+                    <li>8.5 ul Seamless cloning Master Mix</li>
+                    <li>3 ul linear PSB1C3 plasmid</li>
+                    <li>2 ul tRNA </li>
+                    <li>6.5 ul ddH2O
+                      <br>Incubate at 50℃ for 60min.</li>
+                  </ul>
+                  <h5>Seamless Clone of Anchor Plasmid</h5>
+                  <p>Protocol:</p>
+                  <ul>
+                    <li>8.5 ul Seamless cloning Master Mix</li>
+                    <li>3 ul linear PSB1C3 plasmid</li>
+                    <li>1 ul T7 promoter</li>
+                    <li>1 ul COMPX</li>
+                    <li>6.5 ul ddH2O
+                      <br>Incubate at 50℃ for 60min.</li>
+                  </ul>
+                  <h5>Transformation of tRNA Plasmid &amp; Anchor Plasmid</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>4000rpm centrifuge for 3min, discard 500ul supernate.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-02 00:00"><span>2015</span> <span>2/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Plasmid Extraction of C0061</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg. </p>
+                  <h5>PCR of cheZ and OprF from PAO1</h5>
+                  <p>Yesterday, we may extract cheZ from PAO 1, though with a lot of primer dimer. Today, DMSO will be implemented trying to eliminate these fragements.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Taq buffer with KCl</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>25.85</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.65</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>2.5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Taq buffer with KCl</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Opr-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>OprF-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>25.85</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.65</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>2.5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min 30 s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR Purification of T7</h5>
+                  <ol>
+                    <li>Add Buffer B3 in 5 times volumn of PCR product.</li>
+                    <li>Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: OprF, cheZ</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX &amp; PSB1C3-T7-tRNA</h5>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R or tRNA-R Primer</li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p><em>No Result.</em></p>
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX &amp; PSB1C3-T7-tRNA</h5>
+                  <p>Conduct the PCR again.</p>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R or tRNA-R Primer</li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p><em>No Result.</em></p>
+                  <h5>PCR Purification of tRNA &amp; COMPX</h5>
+                  <ol>
+                    <li>Add Buffer B3 in 5 times volumn of PCR product.</li>
+                    <li>Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>Result
+                    <br>tRNA 26ng/ul, COMPX 24ng/ul.</p>
+                  <h5>Transformation of tRNA Plasmid &amp; Anchor Plasmid</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>4000rpm centrifuge for 3min, discard 500ul supernate.</li>
+                    <li>Spread half of the bacteria culture on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-03 00:00"><span>2015</span> <span>3/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX &amp; PSB1C3-T7-tRNA</h5>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R or tRNA-R Primer</li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of OprF from PAO1</h5>
+                  <p>Yesterday, we may extract cheZ from PAO 1, though with a lot of primer dimer. Today, DMSO will be implemented trying to eliminate these fragements.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Taq buffer with KCl</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Opr-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>OprF-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1 Solution</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>28.35</td>
+                      </tr>
+                      <tr>
+                        <td>MgCl2</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.65</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>2.5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of C0061</h5>
+                  <p>material(volume 100uL):
+                    <br>• 6 ul MgCl2
+                    <br>• 71 ul ddH2O
+                    <br>• 4 ul Primer F
+                    <br>• 4 ul Primer R
+                    <br>• 2 ul dNTP
+                    <br>• 1 ul Taq DNA polymerase
+                    <br>• 10 ul 10*PCR Buffer
+                    <br>• 2 ul C0061
+                    <br>PCR cycle including:
+                    <br>1 Preheat: 94 degree celcius, 5min
+                    <br>2 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
+                    <br>3 Elongation for whole at 72 degree celcius 1min
+                    <br>4 4 celcius degree for preserveation.</p>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: R0061
+                    <br>Preparation:
+                    <br>• Check out whether ethyl alcohol is added into Wash Solution.
+                    <br>• Check out whether Buffer B2 has sediment.
+                    <br>• Adjust the thermostat water bath at 50 degree centigrade.
+                    <br>Procedure:
+                    <br>5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
+                    <br>6 Measure out the weight of tubes of gel.
+                    <br>7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
+                    <br>8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
+                    <br>9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
+                    <br>10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
+                    <br>11 Do the step 5 again.
+                    <br>12 Centrifuge the empty columns at 9000xg about 1min.
+                    <br>13 Open the columns and air them about 10 mins.
+                    <br>14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.</p>
+                  <h5>PCR for gRNA-AcrB and gRNA-EmrE</h5>
+                  <p>material:
+                    <br>gRNA-AcrB plasmid(280.7 ng/uL)
+                    <br>gRNA-EmrE plasmid(222.4 ng/uL)</p>
+                  <p>PCR protocol:</p>
+                  <p>For gRNA-AcrB and gRNA-EmrE, primers including gRNA-F, gRNA-R, protocol as following:</p>
+                  <ul>
+                    <li>10μl Taq Master Mix</li>
+                    <li>1μl gRNA-F Primer</li>
+                    <li>1μl gRNA-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>6.75μl ddH2O</li>
+                    <li>1μl gRNA-AcrB(or gRNA-EmrE) Plasmid</li>
+                  </ul>
+                  <p>For gRNA, Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>gRNA-F</td>
+                        <td>GCGTCTAGATTGACAGCTAGCTCAGTAGGTATAAT</td>
+                      </tr>
+                      <tr>
+                        <td>gRNA-R</td>
+                        <td>TAGTAGGTCGTATTAAAAAAAAGCACCGAC</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Electrophoresis Characterization</h5>
+                  <p>gRNA-AcrB and gRNA-EmrE
+                    <br><img src="https://attachments.tower.im/tower/6e9684603ad8480db16cb6f13b1cc8da?version=auto&amp;filename=2015.08.03%20PCR%20of%20gRNA%20ArcB%20EmrE.Tif" alt="图片名称"></p>
+                  <h5>Gel Extraction of gRNA-AcrB and gRNA-EmrE</h5>
+                  <p>Material including: gRNA-AcrB and gRNA-EmrE</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>the concentration as following:
+                    <br>gRNA-AcrB: 44 ng/uL
+                    <br>gRNA-EmrE: 41 ng/uL</p>
+                  <h5>PCR for T7-tet Promoter</h5>
+                  <p>PCR protocol:</p>
+                  <p>For T7-tet promoter, primers including T7-tet-F, T7-tet-R, protocol as following:</p>
+                  <ul>
+                    <li>10μl Taq Master Mix</li>
+                    <li>1μl T7-tet-F Primer</li>
+                    <li>1μl T7-tet-R Primer</li>
+                    <li>1μl template-F Primer</li>
+                    <li>1μl template-R Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>5.75μl ddH2O</li>
+                  </ul>
+                  <p>Use template-F Primer and template-R Primer as templates</p>
+                  <p>For T7-tet, Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>template-F</td>
+                        <td>taatacgactcactatgatagaaaagaggagaaaatc</td>
+                      </tr>
+                      <tr>
+                        <td>template-R</td>
+                        <td>gattttctcctcttttctatcatagtgagtcgtatta</td>
+                      </tr>
+                      <tr>
+                        <td>T7-tet-F</td>
+                        <td>Ttaatacgactcactatgatagaaaagaggag</td>
+                      </tr>
+                      <tr>
+                        <td>T7-tet-R</td>
+                        <td>gtatttcttatccattccgattttctcctcttttctat</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Electrophoresis Characterization</h5>
+                  <p>T7-tet Promoter
+                    <br><img src="https://attachments.tower.im/tower/89c5696e2b4b4bd29e2bedc5b406c80b?version=auto&amp;filename=2015.08.03%20PCR%20of%20T7-tet.Tif" alt="图片名称"></p>
+                  <h5>Gel Extraction of T7-tet Promoter</h5>
+                  <p>Material including: T7-tet Promoter</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>the concentration as following:
+                    <br>T7-tet: 30 ng/uL</p>
+                  <h5>Seamless Clone of cheZ Plasmid and Circuit Containing E0040</h5>
+                  <p>Protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Fragements</th>
+                        <th>Length(bp)</th>
+                        <th>Mass Required(ug)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2070</td>
+                        <td>41.4</td>
+                      </tr>
+                      <tr>
+                        <td>T7 Promoter</td>
+                        <td>46</td>
+                        <td>9.2</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ</td>
+                        <td>683</td>
+                        <td>13.66</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>143</td>
+                        <td>2.86</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>3 ul</td>
+                      </tr>
+                      <tr>
+                        <td>T7 Promoter</td>
+                        <td>1ul(+3 folds ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>cheZ</td>
+                        <td>1ul(+1 fold ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>1ul(+9 folds ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>5.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Fragements</th>
+                        <th>Length(bp)</th>
+                        <th>Mass Required(ug)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2070</td>
+                        <td>41.4</td>
+                      </tr>
+                      <tr>
+                        <td>R0061</td>
+                        <td>30</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>E0040</td>
+                        <td>720</td>
+                        <td>14.4</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>143</td>
+                        <td>2.86</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>3 ul</td>
+                      </tr>
+                      <tr>
+                        <td>R0061</td>
+                        <td>1ul(+20 fold ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>E0040</td>
+                        <td>1ul(+1 fold ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>1ul(+9 folds ddH2O)</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>5.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>PCR for Cas9-PART I and Cas9-PART II</h5>
+                  <p>material:
+                    <br>Cas9 plasmid(511.3 ng/uL)</p>
+                  <p>PCR protocol:</p>
+                  <p>For Cas9-PART I and Cas9-PART II, primers including Cas9-1-F, Cas9-1-R,Cas9-2-F, Cas9-2-R, protocol as following:</p>
+                  <ul>
+                    <li>10μl Taq Master Mix</li>
+                    <li>1μl Cas9-1-F(or Cas9-2-F) Primer</li>
+                    <li>1μl Cas9-1-R(or Cas9-2-R) Primer</li>
+                    <li>0.25μl Taq DNA Polymerase</li>
+                    <li>6.75μl ddH2O</li>
+                    <li>1μl Cas9 Plasmid</li>
+                  </ul>
+                  <p>For Cas9, Primers produced from Sangon Biotech Co.,Ltd.</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primer</th>
+                        <th>Sequence(5'-3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Cas9-1-F</td>
+                        <td>ggaatggataagaaatactcaataggcttag</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-1-R</td>
+                        <td>gctgtttcatcaccttatcatcaaaga</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-2-F</td>
+                        <td>gataaggtgatgaaacagcttaaacgtc</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-2-R</td>
+                        <td>ctactagtgggaccattcaaaacagcatagc</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5min)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Electrophoresis Characterization</h5>
+                  <p>Cas9-PART I and Cas9-PART II
+                    <br><img src="https://attachments.tower.im/tower/0ada4b89e8364446a0b929a9c6fb6076?version=auto&amp;filename=2015.08.04%20PCR%20of%20CAS9%20Part%20I%20II.Tif" alt="图片名称"></p>
+                  <h5>Gel Extraction of Cas9-PART I and Cas9-PART II</h5>
+                  <p>Material including: Cas9-PART I and Cas9-PART II</p>
+                  <p>Preparation:</p>
+                  <ol>
+                    <li>Check out whether ethyl alcohol is added into Wash Solution. </li>
+                    <li>Check out whether Buffer B2 has sediment. </li>
+                    <li>Adjust the thermostat water bath at 50 degree centigrade.</li>
+                  </ol>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers. </li>
+                    <li>Measure out the weight of tubes of gel. </li>
+                    <li>Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins. </li>
+                    <li>Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2. </li>
+                    <li>After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s. </li>
+                    <li>Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution. </li>
+                    <li>Do the step 5 again. </li>
+                    <li>Centrifuge the empty columns at 9000xg about 1min. </li>
+                    <li>Open the columns and air them about 10 mins. </li>
+                    <li>Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min. </li>
+                    <li>Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.</li>
+                  </ol>
+                  <p>the concentration as following:
+                    <br>Cas9-PART I : 31 ng/uL
+                    <br>Cas9-PART II: 16 ng/uL</p>
+                  <h5>Seamless Clone of tRNA Plasmid</h5>
+                  <p>Protocol:</p>
+                  <ul>
+                    <li>8.5 ul Seamless cloning Master Mix</li>
+                    <li>3 ul linear PSB1C3 plasmid</li>
+                    <li>1 ul tRNA </li>
+                    <li>7.5 ul ddH2O</li>
+                  </ul>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Seamless Clone of Anchor Plasmid</h5>
+                  <p>Protocol:</p>
+                  <ul>
+                    <li>8.5 ul Seamless cloning Master Mix</li>
+                    <li>3 ul linear PSB1C3 plasmid</li>
+                    <li>1 ul T7 promoter</li>
+                    <li>1 ul COMPX</li>
+                    <li>6.5 ul ddH2O</li>
+                  </ul>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Transformation of tRNA Plasmid &amp; Anchor Plasmid</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-04 00:00"><span>2015</span> <span>4/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR for micF and C0061</h5>
+                  <p>Protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingradients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>10X PCR buffer</td>
+                        <td>8</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1.6</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>micF-luxI-R</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>Top10 Solution</td>
+                        <td>8</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>53.9</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingradients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>10X PCR buffer</td>
+                        <td>8</td>
+                      </tr>
+                      <tr>
+                        <td>dNTPs</td>
+                        <td>1.6</td>
+                      </tr>
+                      <tr>
+                        <td>C0061-F</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>C0061-B0015-R</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>8</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>53.9</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30s)</li>
+                    <li>Elongation for whole at 72 degree celcius 7 min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Seamless Clone of circuit I</h5>
+                  <p>material:
+                    <br>pSB1C3(XbaI&amp;SpeI digest) 2062bp 7.1ng/uL
+                    <br>micF 402bp 58.7ng/uL
+                    <br>C0061 656bp 27.4ng/uL
+                    <br>B0015 142bp 7.1ng/uL
+                    <br>seamless clone kit</p>
+                  <p>protocol:
+                    <br>pSB1C3 12.4uL
+                    <br>micF 0.5uL
+                    <br>C0061 1.73uL
+                    <br>B0015 1.46uL
+                    <br>seamless clone mix 8.5uL</p>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Transformation of circuit I</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 3min.</li>
+                    <li>Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-05 00:00"><span>2015</span> <span>5/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Seamless Clone of circuit I</h5>
+                  <p>material:
+                    <br>pSB1C3(XbaI&amp;SpeI digest) 2062bp 7.1ng/uL
+                    <br>micF 402bp 58.7ng/uL
+                    <br>C0061 656bp 27.4ng/uL
+                    <br>B0015 142bp 7.1ng/uL
+                    <br>seamless clone kit</p>
+                  <p>protocol:
+                    <br>pSB1C3 12.4uL
+                    <br>micF 0.5uL
+                    <br>C0061 1.73uL
+                    <br>B0015 1.46uL
+                    <br>seamless clone mix 8.5uL</p>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Transformation of circuit I</h5>
+                  <p>Protocol:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 3min.</li>
+                    <li>Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                  <h5>Transformation of pSB1C3</h5>
+                  <p>protocol:</p>
+                  <ol>
+                    <li>Add 2ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 3min.</li>
+                    <li>Add 500ul LB culture(no antibiotics), shaking at 37℃ for 30min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                  <h5>Double digest of pSB1C3</h5>
+                  <p>protocol:
+                    <br>pSB1C3 plasmid solution 10ul
+                    <br>XbaI 1ul
+                    <br>SpeI 1ul
+                    <br>Tango.buffer 2ul
+                    <br>ddH2O 6ul</p>
+                  <p>incubate at 37℃ for 2 hours.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-06 00:00"><span>2015</span> <span>6/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX &amp; PSB1C3-T7-tRNA</h5>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R or tRNA-R Primer</li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Colonial PCR of PSB1C3-T7-cheZ &amp; PSB1C3-R0051-E0040-B0015</h5>
+                  <p>Protocol(volume 13.5ul)</p>
+                  <ul>
+                    <li>0.25μl Primer 1</li>
+                    <li>0.25μl Primer 2</li>
+                    <li>6.5μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>5.5μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Overlap Circuit of gRNA-Cas9</h5>
+                  <p><img src="https://attachments.tower.im/tower/ac422e68e5eb4723809112a98b088e59?version=auto&amp;filename=PSB1C3-gRNA-Cas9.PNG" alt="图片名称"></p>
+                  <h5>Overlapping Fragments</h5>
+                  <p>In order to ligate the fragements gRNA-ArcB or gRNA-EmrE, T7-tet, Cas9-PART I and Cas9-PART II, we need to make the equal mole proportion.</p>
+                  <p>The mole of DNA molecule is calculated in the following ways:</p>
+                  <p><img src="http://chuantu.biz/t2/11/1437963054x-1566638177.gif" alt="图片名称"></p>
+                  <p>among which, c is the concentration of DNA, V is the volumn required and n is the length of DNA. Because of the same molar mass of As, Gs, Cs and Ts, computation of same molar mass is omitted.</p>
+                  <p>According to our sample extracted from PCR and calculation, ingradients characterize as below:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Concentration(ng/uL)</th>
+                        <th>Length(bp)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>gRNA-ArcB</td>
+                        <td>44</td>
+                        <td>162</td>
+                      </tr>
+                      <tr>
+                        <td>gRNA-EmrE</td>
+                        <td>41</td>
+                        <td>162</td>
+                      </tr>
+                      <tr>
+                        <td>T7-tet</td>
+                        <td>30</td>
+                        <td>56</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART I</td>
+                        <td>31</td>
+                        <td>1954</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART II</td>
+                        <td>16</td>
+                        <td>2358</td>
+                      </tr>
+                      <tr>
+                        <td>Total</td>
+                        <td></td>
+                        <td>4530</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>The theoratical volumn proportion is: </p>
+                  <ul>
+                    <li>gRNA-ArcB-T7-tet-Cas9-PART I-Cas9-PART II : 2: 1: 31: 64</li>
+                    <li>gRNA-EmrE-T7-tet-Cas9-PART I-Cas9-PART II: 2: 1: 31: 64</li>
+                  </ul>
+                  <p>Firstly, to ligate these parts, we need 10 cycles without primer:
+                    <br>for circuit gRNA-ArcB-T7-tet-Cas9-PART I-Cas9-PART II:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>gRNA-ArcB</td>
+                        <td>0.2</td>
+                      </tr>
+                      <tr>
+                        <td>T7-tet</td>
+                        <td>0.1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART I</td>
+                        <td>3.1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART II</td>
+                        <td>6.4</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP(+MgCl2)</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>4.95</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for circuit gRNA-EmrE-T7-tet-Cas9-PART I-Cas9-PART II:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>gRNA-EmrE</td>
+                        <td>0.2</td>
+                      </tr>
+                      <tr>
+                        <td>T7-tet</td>
+                        <td>0.1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART I</td>
+                        <td>3.1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-PART II</td>
+                        <td>6.4</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP(+MgCl2)</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>1.25</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>4.7</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 6.5min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <p>Then, we extracted 5 ul as template, and 15 ul adding with primer:
+                    <br>Primers were synthesized from <em>Sangon Biotech. Co.,Ltd.</em></p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Primers</th>
+                        <th>Sequence(5' to 3')</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>gRNA-F</td>
+                        <td>GCGTCTAGATTGACAGCTAGCTCAGTAGGTATAAT</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-2-R</td>
+                        <td>CTACTAATGGGACCATTCAAAACAGCATAGC</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>5ul-template protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Template</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Taq Polymerase</td>
+                        <td>0.25</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP(+MgCl2)</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>1.25</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>7.5</td>
+                      </tr>
+                      <tr>
+                        <td>gRNA-F</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-2-R</td>
+                        <td>1</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>15ul-protocol:</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Name</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Solution</td>
+                        <td>15</td>
+                      </tr>
+                      <tr>
+                        <td>gRNA-F</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Cas9-2-R</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>DMSO</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>10X buffer</td>
+                        <td>2</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 6.5min)</li>
+                    <li>Elongation for whole at 72 degree celcius 1min</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h2 id="aug-6th-2015">Aug 6th, 2015</h2>
+                  <h5>Plasmid Extraction of Circuit 1, B0015, T7</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.</p>
+                  <h5>Enzyme Digestion</h5>
+                  <p>Protocol:
+                    <br>Name Volumn(uL)
+                    <br>pSB1C3 10
+                    <br>XbaI 1
+                    <br>SpeI 1
+                    <br>10* Buffer Tango 2
+                    <br>ddH2O 15</p>
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX</h5>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R </li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Double Digest of T7 Plasmid</h5>
+                  <p>Protocol (Volume 20ul)</p>
+                  <ul>
+                    <li>4ul Tango Buffer</li>
+                    <li>2ul BamHI</li>
+                    <li>2ul SalI</li>
+                    <li>10ul T7 Plasmid</li>
+                    <li>2ul ddH2O
+                      <br>Incubate at 37℃ for 3h.</li>
+                  </ul>
+                  <h5>Colonial PCR of PSB1C3-T7-COMPX</h5>
+                  <p>Conduct the PCR again.</p>
+                  <p>Protocol(volume 20ul)</p>
+                  <ul>
+                    <li>1μl T7-F Primer</li>
+                    <li>1μl COMPX-R </li>
+                    <li>10μl Mix</li>
+                    <li>1μl Bacteria Clone</li>
+                    <li>7μl ddH2O</li>
+                  </ul>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 7min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s), annealing(55 degree celcius, 30s) and elongation(72 degree celcius 1.5 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10min.</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Double Digest of T7 Plasmid</h5>
+                  <p>Protocol (Volume 20ul)</p>
+                  <ul>
+                    <li>4ul Tango Buffer</li>
+                    <li>2ul BamHI</li>
+                    <li>2ul SalI</li>
+                    <li>10ul T7 Plasmid</li>
+                    <li>2ul ddH2O
+                      <br>Incubate at 37℃ overnight.</li>
+                  </ul>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-10 00:00"><span>2015</span> <span>10/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR of C0062</h5>
+                  <p>material(volume 100uL):
+                    <br>• 6 ul MgCl2
+                    <br>• 71 ul ddH2O
+                    <br>• 4 ul Primer F
+                    <br>• 4 ul Primer R
+                    <br>• 2 ul dNTP
+                    <br>• 1 ul pfu DNA polymerase
+                    <br>• 10 ul 10*PCR Buffer
+                    <br>• 2 ul C0062
+                    <br>PCR cycle including:
+                    <br>1 Preheat: 94 degree celcius, 5min
+                    <br>2 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
+                    <br>3 Elongation for whole at 72 degree celcius 1min
+                    <br>4 4 celcius degree for preserveation.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-11 00:00"><span>2015</span> <span>11/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Seamless Clone of pSB1C3-R0010-C0062-B0015</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2 ul</td>
+                      </tr>
+                      <tr>
+                        <td>R0010</td>
+                        <td>0.4ul</td>
+                      </tr>
+                      <tr>
+                        <td>C0062</td>
+                        <td>0.6ul</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>2.4ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>6.1ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50 degree celcius 1 h.</p>
+                  <h5>Seamless Clone of pSB1C3-T7-COMPX</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2ul</td>
+                      </tr>
+                      <tr>
+                        <td>T7</td>
+                        <td>2ul</td>
+                      </tr>
+                      <tr>
+                        <td>COMPX</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>6.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2ul</td>
+                      </tr>
+                      <tr>
+                        <td>T7</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>COMPX</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>14.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50 degree celcius 1 h.</p>
+                  <h5>Transformation of pSB1C3-T7-COMPX</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                  <h5>Enzyme Digestion of pSB1C3-T7-COMPX and pSB1C3-tRNA</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Tango Buffer</td>
+                        <td>2ul</td>
+                      </tr>
+                      <tr>
+                        <td>plasmid</td>
+                        <td>10ul</td>
+                      </tr>
+                      <tr>
+                        <td>Spe I</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>Xba I</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>6ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 37 degree celcius for 2 h.</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-12 00:00"><span>2015</span> <span>12/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Transformation of pSB1C3-R0010-C0062-B0015</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                  <h5>Transformation of pSB3A3</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>Spread on plate with Ampcilin, incubate overnight at 37℃.</li>
+                  </ol>
+                  <h5>Plasmid Extraction of T7</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 4.5 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.</p>
+                  <h5>PCR of OprF from PAO1</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>2XTaq Mix</td>
+                        <td>25</td>
+                      </tr>
+                      <tr>
+                        <td>OprF-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>OprF-Ter-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>PAO1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>17.5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2 min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-13 00:00"><span>2015</span> <span>13/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>Plasmid Extraction of R0062, B0015, SCVE, T7</h5>
+                  <p>Procedure including:
+                    <br>1.Absorb 4.5 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
+                    <br>2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
+                    <br>3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
+                    <br>4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
+                    <br>5.Centrifuge tubes at 12,000xg about 10 mins.
+                    <br>6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
+                    <br>7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
+                    <br>8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
+                    <br>9.Do the step 8 again.
+                    <br>10.Centrifuge the empty columns at 12,000xg about 1 min.
+                    <br>11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.</p>
+                  <h5>PCR of R0062, C0051, E0040, B0015</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 2</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Plasimid Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Pre-annealing of R0051</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>R0051-F1</td>
+                        <td>10</td>
+                      </tr>
+                      <tr>
+                        <td>R0051-R1</td>
+                        <td>10</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate solution in 95 degree celcius 30 min and then used for PCR.</p>
+                  <h5>PCR of R0051, T7-OprF, T7-SCVE, SCVE, micF and soxS</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 2</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Plasimid Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of OprF, micF, SoxS</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 2</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Bacterial Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: R0061, E0040, B0015, T7-OprF
+                    <br>Preparation:
+                    <br>• Check out whether ethyl alcohol is added into Wash Solution.
+                    <br>• Check out whether Buffer B2 has sediment.
+                    <br>• Adjust the thermostat water bath at 50 degree centigrade.</p>
+                  <p>Procedure:
+                    <br>5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
+                    <br>6 Measure out the weight of tubes of gel.
+                    <br>7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
+                    <br>8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
+                    <br>9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
+                    <br>10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
+                    <br>11 Do the step 5 again.
+                    <br>12 Centrifuge the empty columns at 9000xg about 1min.
+                    <br>13 Open the columns and air them about 10 mins.
+                    <br>14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.</p>
+                  <h5>PCR of OprF, SoxS, micF</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 2</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Bactieral Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(<em>Temperature gradient: 48 degree celcius, 50 degree celcius, 53 degree celcius and 55 degree celcius</em> 30s) and elongation(72 degree celcius 2min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: T7-SCVE, SCVE
+                    <br>Preparation:
+                    <br>• Check out whether ethyl alcohol is added into Wash Solution.
+                    <br>• Check out whether Buffer B2 has sediment.
+                    <br>• Adjust the thermostat water bath at 50 degree centigrade.</p>
+                  <p>Procedure:
+                    <br>5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
+                    <br>6 Measure out the weight of tubes of gel.
+                    <br>7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
+                    <br>8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
+                    <br>9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
+                    <br>10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
+                    <br>11 Do the step 5 again.
+                    <br>12 Centrifuge the empty columns at 9000xg about 1min.
+                    <br>13 Open the columns and air them about 10 mins.
+                    <br>14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min</p>
+                  <h5>Enzyme Digestion of pSB1C3</h5>
+                  <p>incubate at 37 degree celcius about 2 h</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Enzyme 1</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Enzyme 2</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>pSB1C3</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>Tango Buffer</td>
+                        <td>4</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>10</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>strategy</th>
+                        <th>Enzyme 1</th>
+                        <th>Enzyme 2</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Strategy 1</td>
+                        <td>EcoRI</td>
+                        <td>PstI</td>
+                      </tr>
+                      <tr>
+                        <td>Strategy 2</td>
+                        <td>EcoRI</td>
+                        <td>SpeI</td>
+                      </tr>
+                      <tr>
+                        <td>Strategy 1</td>
+                        <td>XbaI</td>
+                        <td>PstI</td>
+                      </tr>
+                      <tr>
+                        <td>Strategy 2</td>
+                        <td>XbaI</td>
+                        <td>SpeI</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-14 00:00"><span>2015</span> <span>14/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR of C0061, micF/SoxS-binding C0061</h5>
+                  <p>for C0061</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>C0061-F</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>C0061-B0015-RI</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Plasimid Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for micF-C0061</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>micF-R-BB</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>B0034-C0061-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Plasimid Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>for SoxS-C0061</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>SoxS-R-BB</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>B0034-C0061-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Plasimid Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 2min)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Seamless ligation of pSB1C3-T7-SCVE-B0015</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingradients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>pSB1C3</td>
+                        <td>1.2</td>
+                      </tr>
+                      <tr>
+                        <td>T7-SCVE</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>SCVE</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>Seamless Cloning mix</td>
+                        <td>8.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>to 20</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50 degree celcuis 1 h</p>
+                </div>
+              </li>
+              <li>
+                <time class="cbp_tmtime" datetime="2015-08-15 00:00"><span>2015</span> <span>15/8</span></time>
+                <div class="cbp_tmicon"></div>
+                <div class="cbp_tmlabel">
+                  <h5>PCR of OprF, SoxS</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 1</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Primer 2</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Bacterial Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>PCR of micF-C0061</h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>PCR Buffer</td>
+                        <td>5</td>
+                      </tr>
+                      <tr>
+                        <td>micF-F-BB</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>B0034-C0061-R</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>Bacterial Solution</td>
+                        <td>2.5</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>31.9</td>
+                      </tr>
+                      <tr>
+                        <td>Pfu Polymerase</td>
+                        <td>0.6</td>
+                      </tr>
+                      <tr>
+                        <td>dNTP mix</td>
+                        <td>5</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>PCR cycle including:</p>
+                  <ol>
+                    <li>Preheat: 94 degree celcius, 5min</li>
+                    <li>35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min 30 s)</li>
+                    <li>Elongation for whole at 72 degree celcius 10 mins</li>
+                    <li>4 celcius degree for preserveation.</li>
+                  </ol>
+                  <h5>Gel Extraction</h5>
+                  <p>Material including: micF, pSB1C3
+                    <br>Preparation:
+                    <br>• Check out whether ethyl alcohol is added into Wash Solution.
+                    <br>• Check out whether Buffer B2 has sediment.
+                    <br>• Adjust the thermostat water bath at 50 degree centigrade.</p>
+                  <p>Procedure:
+                    <br>5 Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
+                    <br>6 Measure out the weight of tubes of gel.
+                    <br>7 Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
+                    <br>8 Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
+                    <br>9 After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
+                    <br>10 Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
+                    <br>11 Do the step 5 again.
+                    <br>12 Centrifuge the empty columns at 9000xg about 1min.
+                    <br>13 Open the columns and air them about 10 mins.
+                    <br>14 Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.</p>
+                  <h5>Ligation of pSB1C3-tRNA</h5>
+                  <p>incubate at 16 degree celcius more than 20 h</p>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn(uL)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>T4 DNA ligation Buffer</td>
+                        <td>2</td>
+                      </tr>
+                      <tr>
+                        <td>T4 DNA ligase</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>pSB1C3 digested by EcoRI and PstI</td>
+                        <td>1</td>
+                      </tr>
+                      <tr>
+                        <td>tRNA</td>
+                        <td>6</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>11</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <h5>Seamless Cloning of pSB1C3-micF-C0061-B0015 </h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2 ul</td>
+                      </tr>
+                      <tr>
+                        <td>micF</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>C0061</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>B0015</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>6.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Seamless Cloning of pSB1C3-T7-COMPX </h5>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th>Ingredients</th>
+                        <th>Volumn</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td>Seamless cloning Master Mix</td>
+                        <td>8.5 ul</td>
+                      </tr>
+                      <tr>
+                        <td>linear PSB1C3 plasmid</td>
+                        <td>2 ul</td>
+                      </tr>
+                      <tr>
+                        <td>COMPX</td>
+                        <td>1ul</td>
+                      </tr>
+                      <tr>
+                        <td>ddH2O</td>
+                        <td>8.5ul</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <p>Incubate at 50℃ for 60min.</p>
+                  <h5>Transformation of pSB1C3-micF-C0061-B0015 and pSB1C3-T7-COMPX</h5>
+                  <p>Procedure:</p>
+                  <ol>
+                    <li>Add 4ul clone product to competent cells, 1mix well, incubate on ice for 30min.</li>
+                    <li>Heat in 42℃ bath for 90s. Then put on ice for 5min.</li>
+                    <li>Add 500ul SOC culture, incubate at 37℃ for 45min.</li>
+                    <li>Spread on plate with chloramphenicol, incubate overnight at 37℃.</li>
+                  </ol>
+                </div>
+              </li>
+          </div>
+        </div>
+      </div>
+    </div>
-<h5>What should this page have?</h5>
+    <div id="Raw-data" class="row">
-<ul>
+      <div class="card hoverable">
-<li>Chronological notes of what your team is doing.</li>
+        <div class="col s12 m9">
-<li> Brief descriptions of daily important events.</li>
+        	<div class="card-content">
-<li>Pictures of your progress. </li>
+            Raw-data
-<li>Mention who participated in what task.</li>
+          </div>
-</ul>
+        </div>
+        <div class="col hide-on-small-only m3">
+          <div class="toc-wrapper pinned">
-<h4>Inspiration</h4>
+            <ul class="section table-of-contents">
-<p>You can see what others teams have done to organize their notes:</p>
+              <li>
+                <a href="#Raw-data">Raw-data</a>
-<ul>
+              </li>
-<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+            </ul>
-<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+          </div>
-<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+        </div>
-<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+      </div>
-</ul>
+    </div>
+  </div>
 </div>
 </html>
 {{USTC/footer}}

Difference between revisions of "Team:USTC/Notebook"

Revision as of 07:26, 13 September 2015

Medium preparation

Cell culture

Design protocol

转化

绿脓杆菌接菌

冻存绿脓杆菌

涂板绿脓杆菌

转化

1st Colonial PCR for COMPX

1st Electrophoresis for Colonical PCR

重新转化实验

重新制备感受态细胞

重新对绿脓杆菌涂板

2nd Colonial PCR for COMPX

2nd Electrophoresis for Colonical PCR

3rd Colonial PCR for COMPX

3rd Electrophoresis for Colonical PCR

1st Transformation

4th Colonial PCR for COMPX

4th Electrophoresis for Colonical PCR

1st Plasmid Extraction for BBa K1492002 & pSB1C3

1st Electrophoresis for Plasmid Extraction

绿脓杆菌划线

The steak-plate of Pseudomonas aeruginosa

2nd Plasmid Extraction for BBa K1492002 & pSB1C3

2nd Electrophoresis for Plasmid Extraction

1st PCR for tRNA

1st Electrophoresis for tRNA PCR

Transformation of Four Parts of Plate 4

The Results of Transformation of Jul 13th

Plasmid Extraction of pSB1C3

Transformation of Three Parts and One Plasmid

Plasmid Extraction of Three Parts of Plate 4

Results of transformation

Plasmid Extraction Results

Colonical PCR for micF and SoxS

Characterization of Colonical PCR by Gel Electrophoresis

Colonical PCR for OprF from Psudomonus aeruginosa

Plasmid Extraction of SCVE

Transformation for CRISPR

Characterization of SCVE and OprF by Gel Electrophoresis

Digestion of E0040(EGFP)

PCR of E0040(EGFP)

Characterization of E0040 from Digestion and PCR

Gel Extraction

Transfofmation of Three Parts

Plasmid Extraction of E0040

Colonical PCR for OprF from Psudomonus aeruginosa

Digestion of E0040(EGFP)

Characterization of OprF and E0040

Plasmid Extraction of Cas9(K1218011)

Characterization of Cas9

Digestion of K1218011(Cas9)

Digestion of SCVE

Colonical PCR for OprF from *Psudomonus aeruginosa, micF and SoxS from K-12

Praparation for Competent Cell

Negative Chemotaxis Characterization

Plasmid Extraction of EmrE and ArcB

Transformation of R0051 and C0061

PCR of OrpF

PCR of OprF

OprF for PCR and gel electrophoresisOprF

Preparation of Semi-Solid LB Medium

Plasmid Extraction C0051,B0015 and R0062

PCR of C0051,B0015 and R0062

Preparation Semi-Solid M63 Medium

Colonical PCR for OprF from Psudomonus aeruginosa and SCVE from pUC57

Gel Electrophoresis of OprF, SCVE micF and SoxS

Gel Extraction

Plasmid Extraction of R0062

PCR for OprF and C0061

Gel Extraction of C0062,B0015 and R0062

PCR of OprF

Gel Electrophoresis Characterization

Preparation for Homologous Recombination: Plasimid Digestion

PCR of C0061,B0015 and micF

PCR of C0061,B0015,R0062,R0010 and C0062

Characterization of micF and SoxS by Gel Electrophoresis