Team:USTC/Notebook
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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.
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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.
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。
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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。 -
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。 -
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。
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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：
- 冰上放置感受态细胞2~3min，一共4管
- 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
- 在42℃水浴锅中热激45s
- 冰上放置3min
- 加入900ulLB培养基培养55min
- 离心12000g 15s，放弃上清液，加入200ulLB培养基
- 涂板，板带有氨苄抗性
- 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。
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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：
- 冰上放置感受态细胞3min，一共4管
- 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。
- 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
- 在42℃水浴锅中热激55s
- 冰上放置3min
- 加入到5mL的氯霉素抗性的液体培养基。
- 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.
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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℃.
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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。
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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
- 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 - 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
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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 R0051Plasmid 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 319the 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
- 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 - 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 platePlasmid 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 - the plasmid going to be transformed:
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Results of transformation
R0051 no colony
R0062 have single colony
B0015 have single colony
pSB1C3 have single colony and the colony is redPlasmid 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.
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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
The result illustrated the success of PCR -
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
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
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.
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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
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
转化成功，明天写实验结果讨论 -
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.
-
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/ulTransformation 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 4No.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 platePCR 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
-
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
-
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/uLPCR 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-foldsPrimers 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
OprF and SCVE
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.
-
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/uLPCR 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
OprF
That was relatively successully extracted OprF from colonical PCR.R0062
-
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/uLForB0015 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
-
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 R0010:243bpCharacterization of micF and SoxS by Gel Electrophoresis
micF were successfully PCRed.Characterization of pSB1C3 and SoxS by Gel Electrophoresis
-
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:
- 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.
Characterization of B0015 and R0051 by Gel Electrophoresis
-
Overlap Circuit
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:
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/uLFor 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. -
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 polymerasePCR 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.
-
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 . -
Negative Chemotaxis Characterization
Done by Wang Luyao
7.30Term 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:
- 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.
-
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 SolutionPrimers 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 -
-
Negative Chemotaxis Characterzation
Done by Wang Luyao
Term 3 results
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:
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 SolutionPrimers 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 Solutionprotocol:
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℃.
-
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℃.
-
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
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/uLPCR 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
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/uLSeamless 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
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/uLSeamless 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℃.
-
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 kitprotocol:
pSB1C3 12.4uL
micF 0.5uL
C0061 1.73uL
B0015 1.46uL
seamless clone mix 8.5uLIncubate 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℃.
-
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 kitprotocol:
pSB1C3 12.4uL
micF 0.5uL
C0061 1.73uL
B0015 1.46uL
seamless clone mix 8.5uLIncubate 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 6ulincubate at 37℃ for 2 hours.
-
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
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:
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 15Colonial 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.
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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. -
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.
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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.
-
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 minEnzyme 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 -
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
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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℃.