Difference between revisions of "Team:Tianjin/Notebook"

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         <tr>
 
         <tr>
 
             <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 
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 +
 
                 <p style="text-align:center">
 
                 <p style="text-align:center">
 
                     <span style="font-size:  19px;font-family:&#39;Arial&#39;,sans-serif;color:black">XbaI</span>
 
                     <span style="font-size:  19px;font-family:&#39;Arial&#39;,sans-serif;color:black">XbaI</span>
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<br>
 
<br>
 
N is the medium in the column after being washed by MCAC 100
 
N is the medium in the column after being washed by MCAC 100
 +
 
<br>
 
<br>
 
O is the MCAC 200 flowing through the Ni column
 
O is the MCAC 200 flowing through the Ni column
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Revision as of 01:57, 19 September 2015

内页

Receive standard plasmid

1. Gain standard plasmid

2. Transformation:

Result: After 16~24h overnight culturing, sJanus, sJanus-m and inJanus grow colonies about one microns in diameter, evenly distributed. But inJanus-m’ colonies are relatively rare and small. Try to going on to check out

3. Strain reservation and plasmid extraction

Results: Four tube plasmid concentration is about 100 ng/uL, suitable for next steps

4. Striction Digest

I choose Xba I and Pst enzyme

5. Electrophoresis

Results: We have strips of about 2000. We can basically ensure correct standard transformation

6, DNA gel extraction   

Results: Four tube plasmid concentration is 4.9, 6.0, 9.4, 6.2 ng/uL.

 

Prepare aimed protein DNA

1. Design primers.
2. PCR 50 uL system:


ddH2O

33uL

10Xbuffer

5uL

dNTP

5uL

Template

1uL

Enzyme

1uL

Primer

2.5+2.5u

Put in PCR system, set up annealing temperature, recycle and start PCR.

3. Electrophoresis

Results: Strips of a 200 is aimed proteins. At first time, we set,58,60,62,but we can’t get aimed band, just a very bright band much smaller than aimed DNA. We think its primer dimer, then we set 50,55,60at second time. We can get aimed DNA from 55-60.

4. DAN extraction
5. Restriction enzyme digestion
6. Electrophoresis
7. DAN extraction

Use the same enzyme as one in aimed DNA.

 

Connection 

1. Connection
2. Transformation
3. Plasmid extraction The concentration is 60, 43, 70, 83 ng/mL
4. Enzyme digestion   
5. Electrophoresis

Result: I repeat 4 times. In first time there aren’t any 200 bond. I suspect it’s something wrong in enzyme digest. And then I use four pair of enzymes to cut it. There is just no 200 bond. At the fourth time, I choose to extend digestion time and smaller enzymes of XbaI and PstI. Then we have two stripes: about 200 and 2000 molecular weight.

6. Sequencing:

Almost correct except some wrong sequence in the front of the aimed proteins.

 


17.05.15

·Transformation FsC-pSB1C3 with DH5α

19.05.15

·Inoculation of LB-media with DH5α_FsC-pSB1C3; Overnight incubation at 37°C

20.05.15

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of DH5α_FsC-pSB1C3 using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

(1): 24.1 ng/µl
(2): 33.4 ng/µl
(3): 28.6 ng/µl

26.05.15

·PCR of FsC-pSB1C3 for amplification of FsC

In a sterile 0.5ml microfuge tube, mix in the following order:

ddH2O

68μL

10x Pfu buffer with MgSO4

10μL

dNTP(2.5mM)

8μL

Primer F

5μL

Primer R

5μL

Template DNA

2μL

Pfu DNA   polymerase(2.5U/μL)

2μL

Total volume

100μL

Separate into four sterile 0.5ml microfuge tubes to make 25μL as final volume.

The primer F and R showed below:

F: 5’-GAATTCGCGGCCGCTTCTAGATGCTGCCTACTTCTAACCCT-3’

R: 5’-AGAACCTCCACCGCCAGAACCTCCTCCACCAGCAGAACCACGGAC-3’

PCR gradient is 56.4°C, 57.6°C, 60.9°C, 64.4°C.

27.05.15

·Analysis of PCR products by agarose gel electrophoresis. No bands visible.

Possible reason is the template is not correct, so we extract plasmid again from DH5α.

·Inoculation of LB-media with DH5α_FsC-pSB1C3; Overnight incubation at 37°C

28.05.15

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of DH5α_FsC-pSB1C3 using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

(1): 38.4 ng/µl
(2): 62.9 ng/µl

·PCR of FsC-pSB1C3 for amplification of FsC

Improve the system of PCR:

System I:

ddH2O

66μL

10x Pfu buffer with MgSO4

10μL

dNTP(2.5mM)

10μL

Primer F

5μL

Primer R

5μL

Template DNA

2μL

pfu DNA polymerase(2.5U/μL)

2μL

Total volume

100μL

System I:

ddH2O

66μL

10x Pfu buffer with MgSO4

10μL

dNTP(2.5mM)

10μL

DMSO

6μL

Primer F

5μL

Primer R

5μL

Template DNA

2μL

pfu DNA polymerase(2.5U/μL)

2μL

Total volume

100μL

 


Improve the efficiency of PCR by decreasing annealing temperature. Annealing gradient is 50.1°C, 55.4°C, 60.6°C, 64.5°C

29.05.15

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

Figure 1. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a to d are PCR products of System I, Template DNA(1), annealing gradient in an increasing order. e to h are PCR products of System I, Template DNA(2), annealing gradient in an increasing order. i to l are PCR products of System II, Template DNA(1), annealing gradient in an increasing order. m to o are PCR products of System II, Template DNA(2), annealing gradient in an increasing order.

Only the PCR product of System II, Template DNA(1), annealing temperature is 50.1°C has band at 650bp.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product: 8.7ng/µl

·PCR of PCR product for amplification of FsC

Use System II and decrease annealing temperature. Annealing gradient is 48.3°C, 49.1°C, 50.6°C, 52.4°C

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

Figure 2. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a to d are PCR products of FsC.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product: 65.5ng/µl

·PCR for amplification of four types of Jauns (sJanus, sJanus-m, inJanus, inJanus-m)

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

demo.jpg

Figure 3. The result of agarose gel electrophoresis. M is DNA marker (DL2000 DNA Marker). a to d are PCR products of sJanus. e to h are PCR products of sJanus-m. i to k are PCR products of inJanus. l to o are PCR products of inJanus-m.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product:

sJanus: 30.2ng/µl

sJanus-m: 45.5ng/µl

inJanus: 58.4ng/µl

inJanus-m: 64.6ng/µl

15.06.15

·Restriction digest of pSB1C3 using XbaI and PstI

System I

ddH2O

21µl

Plasmid

3µl

Green buffer

3µl

XbaI

1.5µl

PstI

1.5µl

Total   volume

30µl

 


System II

ddH2O

14µl

Plasmid

10µl

Green buffer

3µl

XbaI

1.5µl

PstI

1.5µl

Total volume

30µl

 

Digest at 37°C for 2h.

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 4. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a is digest product in System I. b is digest product in System II. c is control which is not digested by XbaI and PstI.

The bands are not in a right place. So we sequence FsC-pSB1C3. The result is no signal. Thus, we transform plasmid again with DH5αto get a correct plasmid.

08.07.15

·Transformation FsC-pSB1C3 with DH5α

09.07.15

·Inoculation of LB-media with DH5α_FsC-pSB1C3; Overnight incubation at 37°C

10.07.15

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of DH5α_FsC-pSB1C3 using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

(1): 118.4 ng/µl

(2): 100.8 ng/µl

11.07.15

·Restriction digest of pSB1C3 using XbaI and PstI for verification.

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 5. The result of agarose gel electrophoresis. M1 is DNA marker (Trans2K DNA Marker). M2 is DNA marker (Trans8K DNA Marker).a is digest product of FsC-pSB1C3.

·Overlap PCR of fragment FsC and four types Janus for amplification of fusion fragment

Annealing gradient is 58.8°C, 61.1°C, 63.0°C and 64.3°C

·Analysis of Overlap PCR products by agarose gel electrophoresis.

demo.jpgdemo.jpg

demo.jpg


Figure 6. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a to d are Overlap PCR products of FsC-sJanus. e to h are Overlap PCR products of FsC-sJanus-m. i to l are PCR Overlap products of FsC-inJanus. m to o are Overlap PCR products of FsC-inJanus-m. All the annealing gradient is in an decreasing order.

12.07.15

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product:

FsC-sJanus: 92.4ng/µl

FsC-sJanus-m: 67.4ng/µl

FsC-inJanus: 80.5ng/µl

FsC-inJanus-m: 75.3ng/µl

14.07.15

·Verification of Overlap PCR

  PCR of fusion fragment for verification fragment FsC and fragment Janus.

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

demo.jpg


Figure 7. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a to d are PCR products of FsC which use FsC-sJanus, FsC-sJanus-m, FsC-INJANUS, FsC-inJanus-m,as template respectively. e, g, i, k are PCR products of Janus which use FsC-SJANUS, FsC-sJanus-m, FsC-inJanus, FsC-inJanus-m as template respectively. f, h, j, l are PCR products of Janus which use sJanus, sJanus-m, inJanus, inJanus-m as template respectively.

Fragment FsC and four types of Janus can amplify from fusion fragment which means Overlap PCR is successful.

16.07.15

·PCR of four types fusion fragment for amplification of expression fragment

17.07.15

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

demo.jpg

demo.jpg


Figure 8. The result of agarose gel electrophoresis. M is DNA marker (DL2000 DNA Marker). a1 to a4 are PCR products of FsC expression fragment. b1 to b4 are PCR products of FsC-sJanus expression fragment. c1 to c4 are PCR products of FsC -sJanus-m expression fragment. d1 to d4 are PCR products of FsC-inJanus expression fragment. e1 to e4 are PCR products of FsC-inJanus-m expression fragment.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product:

FsC: 84.6ng/µl

FsC-sJanus: 78.3ng/µl

FsC-sJanus-m: 43.8ng/µl

FsC-inJanus: 61.6ng/µl

FsC-inJanus-m: 48.9ng/µl

18.07.15

·Transformation plasmid pET-28a with Trans1-T1

19.07.15

·Inoculation of LB-media with Trans1-T1_ pET-28a; Overnight incubation at 37°C

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of Trans1-T1_ pET-28a using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

(1): 146.7 ng/µl

(2): 139.5ng/µl

20.07.15

·Restriction digest of plasmid pET-28a, FsC expression fragment, FsC-sJanus-m expression fragment, FsC-inJanus-m expression fragment using XhoI and EcoRI

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 9. The result of agarose gel electrophoresis. M1 is DNA marker (Trans8K DNA Marker). M2 is DNA marker (DL2000 DNA Marker). a is restriction digest product of pET-28a. b is restriction digest product of FsC expression fragment. c is restriction digest product of FsC-sJanus-m expression fragment. d is restriction digest product of FsC-inJanus-m expression fragment.

·Ligation of the digested genes (FsC expression fragment, FsC-sJanus-m expression fragment, FsC-inJanus-m expression fragment) into pET-28a.

·Transformation ligation products with DH5α

22.07.15

·Inoculation of LB-media with DH5α_ FsC_pET-28a, DH5α_ FsC-sJanus-m_pET-28a, DH5α_ FsC-inJanus-m_pET-28a; Overnight incubation at 37°C

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of DH5α_ FsC_pET-28a, DH5α_ FsC-sJanus-m_pET-28a, DH5α_ FsC-inJanus-m_pET-28a using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

FsC_pET-28a (1): 108.4 ng/µl

FsC_pET-28a (2): 138.7ng/µl

FsC-sJanus-m_pET-28a (1): 96.8 ng/µl

FsC-sJanus-m _pET-28a (2): 114.5ng/µl

FsC-inJanus-m _pET-28a (1): 103.7 ng/µl

FsC-inJanus-m _pET-28a (2): 125.5ng/µl

·Verification of ligation product by restriction digest.

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 10. The result of agarose gel electrophoresis. M1 is DNA marker (DL2000 DNA Marker). M2 is DNA marker (Trans8K DNA Marker). a1 is restriction digest product of FsC-inJanus-m _pET-28a (2). a2 is restriction digest product of FsC-inJanus-m _pET-28a (1). b1 is restriction digest product of FsC-sJanus-m _pET-28a (2). b2 is restriction digest product of FsC-sJanus-m_pET-28a (1). c1 is restriction digest product of FsC _pET-28a (2). c2 is restriction digest product of FsC _pET-28a (1).

Except FsC-inJanus-m _pET-28a (1), all the ligation products are correct through restriction digest. Sequencing DNA of these three kinds recombinant plasmid.

23.07.15

The templates of Janus have been confused through the result of sequencing DNA. sJanus-m is confused with SJANUS. inJanus is confused with inJanus-m and eventually we only get FsC-sJanus. We restart from PCR of other three types of Janus.

31.07.15

·PCR for amplification of three types of Janus (sJanus-m, inJanus, inJanus-m)

01.08.15

·Analysis of PCR products by agarose gel electrophoresis.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product:

sJanus-m: 57.5ng/µl

INJANUS: 89.4ng/µl

inJanus-m: 77.6ng/µl

·Overlap PCR of fragment FsC and four types Janus for amplification of fusion fragment

·Analysis of Overlap PCR products by agarose gel electrophoresis.

demo.jpg

Figure 11. The result of agarose gel electrophoresis. M is DNA marker (DL2000 DNA Marker). a1 to a4 are Overlap PCR products of FsC-sJanus-m. b1 to b4 are Overlap PCR products of FsC-INJANUS. c1 to c4 are PCR Overlap products of FsC-inJanus-m.

No bands in the b1 to b4 which means the Overlap PCR of FsC-INJANUS are failed.

·Verification of Overlap PCR

  PCR of fusion fragment for verification fragment FsC and fragment Janus.

02.08.15

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

Figure 12. The result of agarose gel electrophoresis. M is DNA marker (Trans2K DNA Marker). a and c are PCR products of FsC which use FsC-sJanus-m, FsC-inJanus-m as template respectively. b and d are PCR products of Janus which uses FsC-sJanus-m, FsC-inJanus-m as template respectively.

Fragment FsC and two types of Janus can amplify from fusion fragment which means Overlap PCR is successful.

·PCR of two types fusion fragment for amplification of expression fragment

·Analysis of PCR products by agarose gel electrophoresis.

demo.jpg

Figure 13. The result of agarose gel electrophoresis. M is DNA marker (DL2000 DNA Marker). a1 to a4 are PCR products of FsC -sJanus-m expression fragment. b1 to b4 are PCR products of FsC-inJanus-m expression fragment.

·PCR product purification using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified PCR product:

FsC-sJanus-m:73.6ng/µl

FsC-inJanus-m: 66.4ng/µl

·Restriction digest of pET-28a, FsC-sJanus-m expression fragment, FsC-inJanus-m expression fragment using XhoI and EcoRI

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 14. The result of agarose gel electrophoresis. M1 is DNA marker (Trans8K DNA Marker). M2 is DNA marker (DL2000 DNA Marker). a is restriction digest product of pET-28a. b is restriction digest product of FsC-sJanus-m expression fragment. d is restriction digest product of FsC-inJanus-m expression fragment.

·PCR product purification using the TIANgel Midi Purification Kit

·Ligation of the digested genes (FsC-sJanus-m expression fragment, FsC-inJanus-m expression fragment) into pET-28a.

·Transformation ligation products with DH5α

03.08.15

·Inoculation of LB-media with DH5α_ FsC-sJanus-m_pET-28a, DH5α_ FsC-inJanus-m_pET-28a; Overnight incubation at 37°C

04.08.15

·Storage the overnight incubated bacteria at -80 with glycerin (final concentration is 20%)

·Miniprep of DH5α_ FsC-sJanus-m_pET-28a, DH5α_ FsC-inJanus-m_pET-28a using the TIANgel Midi Purification Kit

Nanodrop measurements of the purified plasmid:

FsC-sJanus-m_pET-28a (1): 87.4ng/µl

FsC-sJanus-m _pET-28a (2): 95.9ng/µl

FsC-inJanus-m _pET-28a (1): 125.6ng/µl

FsC-inJanus-m _pET-28a (2): 103.4ng/µl

·Verification of ligation product by restriction digest.

·Analysis of restriction digest by agarose gel electrophoresis.

demo.jpg

Figure 15. The result of agarose gel electrophoresis. M1 is DNA marker (DL2000 DNA Marker). M2 is DNA marker (Trans8K DNA Marker). a is restriction digest product of FsC-sJanus-m_pET-28a (1). b is restriction digest product of FsC-sJanus-m _pET-28a (2). c is restriction digest product of FsC-inJanus-m _pET-28a (1). d is restriction digest product of FsC-inJanus-m_pET-28a (2).

Two ligation products (FsC-sJanus-m_pET-28a (1), FsC-inJanus-m _pET-28a (2)) are correct through restriction digest. Sequencing DNA of these two kinds recombinant plasmid.

05.08.15

The results of sequencing DNA are correct.

07.08.15

·Transformation FsC _pET-28a, FsC-sJanus_pET-28a, FsC-sJanus-m_pET-28a with BL21.

 


We mixed 200ng of the target gene sequence-Thc_Cut1, which is synthesized by IDT Company, with 50ul ddH20, making the final concentration up to 4ng/μl. And then, we divided them into five eppendorf tubes equally. We confirmed the right of sequence of Thc_Cut1 by PCR.

demo.jpg

We made sure that 58.8 is suitable for amplifying by the PCR of Thc_Cut1 which we have done. Next, we amplified the gene of Thc_Cut1, sJanus and its mutant sJanus-m by PCR. In this process, we didn’t do pre-experiment because of the experience of other groups. The temperature of PCR ranges from 55 centigrade to 65 centigrade. The following pictures of gel show that we got the target sequence successfully. (This is the standardized part.)


demo.jpg

demo.jpg


Today, we got the primers of inJanus and its mutant inJanus-m. In the experiment, we constructed a gradient of temperature, ranging from 55.7 centigrade to 63.0 centigrade to search for the best condition. The picture of them show that all of them is suitable, but the temperature of 56.9 is the best. And then, we make the target sequence recycled. (This is the standardized part.)

demo.jpgdemo.jpg

We linked the Thc_Cut1 with sJanus in the third step of overlap. The following picture shows the reagents we used and the exact amount we added in this system. The condition is the same to the process of our getting the gene of Thc_Cut1- sJanus-m. Using the method of PAGE, we found our target gene sequence and make them recycled.

Overlap system (100μl)

10×Buffer

10μl

dNTPS

10μl

Thc­_Thc_Cut1   standardized fragment

5μl

Hydrophobin standardized   fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

After the process of pre-PCR, we put the forward and reverse primer to this system, each for 4μl, and then continued the PCR as usual.


demo.jpg

demo.jpg

We confirmed that the final gene sequence is right by PCR. In this process, we use the target sequence we got in above steps as the template DNA. And then, we added the primer and the reverse of Thc_Cut1, sJanus and sJanus-m in the PCR system. Using method of PAGE, we can confirmed that these two gene have been linked together correctly. The following picture shows the reagents we used and the exact amount we added in this system.

PCR system10μl

10×Buffer

1μl

dNTPS

1μl

Forward primer

0.4μl

Reverse primer

0.4μl

Overlap fragment

0.4μl

Pfu enzyme

0.2μl

ddH2O

6.6μl

 

 demo.jpgdemo.jpg

We used enzymes of XbaI and PstI to digest the product PCR and plasmid, and restored them in the -20 centigrade.

restriction enzyme digestion system (30μl)

ddH2O

10μl

ddH2o

16μl

10×Buffer

3μl

10×Buffer

3μl

Xba1

1.5μl

Xba1

1μl

Pst1

1.5μl

Pst1

1μl

DNA(plasmid)

14μl

DNA(PCR fragment)

10μl

 

We used T4 DNA ligase to connect the gene coding Thc_Cut1- sJanus with the plasmid of pSB1C3. In this experiment, the amount of product is about 5 times of plasmid.

 The following picture shows the reagents we used and the exact amount we added in the system of connection.

connection system

10×Buffer

2 μl

T4  ligase

1 μl

plasmid

X μl

fragment

5x μl

 

There are bacterial colony in the selective medium containing chloramphenicol. We selected three of them everyone, added into the tubes containing LB. And then, we incubated them in the shaker at the temperature of 37 centigrade overnight.

 When incubated overnight, we measured OD to judge whether the cells could be used to extract plasmid. We selected the suitable ones, and extracted the plasmid. And then, we used enzymes of Xba1 and Pst1 to digest some of them. The total volume of enzyme digest is 10μl. Using the method of PAGE, we know that the plasmid connected with Thc_Cut1- sJanus-m may be right. We sent our sample to company, using the technique of Solexa to check it.

demo.jpg

We used the standardized gene as the template DNA to amplify the gene of expression. In this process, we also got the sequence of Thc_Cut1 which can be transferred and secret. The following picture of gel shows that we have got our target sequence successfully. The temperature of PCR is 60 centigrade.

demo.jpg

We added 5μl of the bacterial containing the plasmid of pET28a(+) to 5mL of LB, which contains 5μl of ampicillin. And then, we incubated them at the shaker overnight.

 We exacted the plasmid and used enzymes of EcoR1 and Xho1 to digest it. In this process, the total volume of this system is 20μl. We also did the same to the target sequence and the total volume is 30μl. After finishing above all, we used gel electrophoresis to test and make the gene recycled.

 We connected the gene coding Thc_Cut1, Thc_Cut1- sJanus, Thc_Cut1- sJanus-m, Thc_Cut1- inJanus and Thc_Cut1- inJanus-m with plasmid of pET28a(+). The reagents and amount we used are same to above. The total volume of this system is 20μl. Finally, we transferred them into competent cells and incubated overnight.

 When incubated overnight, there are bacterial colonies in the selective medium contain ampicillin. We selected two of them and added into LB. Finally, we incubated them overnight. In this process, we can judge whether the plasmids we constructed are right by enzyme digesting.

 When incubated overnight, we measured OD to judge whether the cells could be used to extract plasmid. We selected the suitable ones, and extracted the plasmid. And then, we used enzymes of EcoR1 and Xho1 to digest some of them. The total volume of enzyme digest is 10μl. Using the method of PAGE, we know that the plasmid connected with Thc_Cut1 and Thc_Cut1- sJanus-m may be right. We sent our sample to company, using the technique of Solexa to check them.


5.27 Get the PCR products of four basic parts.

demo.jpg

Figure 1. We got the PCR products of injanus, sjanus and sjanus-m. However, for some unknown reason, we didn’t get the PCR products of injanus-m. Although we had set temperature gradient as figure 1 showed.

7.11 overlap PCR

demo.jpg

Figure 2.  In order to get the final parts which used to express protein. We did overlap PCR to get LC-sjanus, LC-injanus-m and LC-injanus-m. The detailed protocols can be seen as follow.

PCR system100μl

Buffer

10μl

dNTPS

8μl

LC   fragment

3μl

sJanus   fragment

3μl

Pfu   enzyme

2μl

ddH2O

70μl

Add two kinds of primer (2μl) after 5 cycle.

5 cycles

95

5min

95

30s

158.8

261.1

364.3

30s

72

2min30s

72

10min

4

Forever

   35 cycles

95

5min

95

30s

158.8

261.1

364.3

30s

72

2min30s

72

10min

4

Forever

Overlap PCR of LC-sjanus( used in PSB1C3)

demo.jpg

Figure 3.  We tried to do overlap PCR of LC-sjanus used in PSB1C3 first. Then we used this as template DNA to PCR LC-sjanus used in expression.

In this step, we got very light electrophoresis strip the length of which is about 1200 bp.

7.12   PCR of LC-sjanus used in expression

demo.jpg

Figure 4.  Failed. We didn’t get the product. The reason may be that the specificity of the primers was not high.

7.13 Restriction digest of LC-injanus, LC-injanus-m and LC-sjanus using EcoR1 and Xhol1

demo.jpg

Figure 5.  Restriction digest of LC-injanus, LC-injanus-m and LC-sjanus using EcoR1 and Xhol1.

7.15 verify our PCR products

demo.jpg

Figure 6.  In order to prove that the parts we got used in expression is correct, we used LC-injanus, LC-injanus-m, LC-sjanus and LC-sjanus-m as templates to pcr LC and other four basic parts: injanus, injanus-m, sjanus, sjanus-m. The result was partially successful. It is very clear in this figure that all parts have LC, but LC-injanus and LC-injanus-m do not have their other part. It may because that our PCR system 10μis too small.

7.16  verify our parts again

demo.jpg

Figure 7.  This figure shows clearly that LC-sjanus-m, LC-injanus and LC-injanus-m are all correct.

7.17  Overlap PCR  LC-sJanus  (compared with other group adding DMSO )

 

demo.jpg

Figure 8.  Failed. We can not see any differences.

7.18  change primers

demo.jpg

Figure 9.  We changed primers to do overlap pcr but failed.

7.22 LC-sJanus overlap pcr

demo.jpg

Figure 10.  We changed our pcr system from 50μ to 25μ, and changed cycle times from 5 to 10.

7.23 Colony PCR of LC-sjanus-m and LC-injanus-m after transformation.

demo.jpg

Figure 11.  Colony PCR. Finally four lanes had light electrophoresis strips. But after enzyme digestion showed that they were all false positive.

7.24  enzyme digestion of LC-sjanus-m and LC-injanus-m after transformation.

demo.jpg

Figure 12.  Failed.

Verification PCR products of LC-sjanus.

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Figure 13.  We verified the correction of LC-sjanus pcr products. The result showed that it was correct.

Amplification PCR of LC-sjanus

demo.jpg

Figure 14.  We successfully amplified LC-sjanus although the electrophoresis strips were light.

7.28 Enzyme digest LC-sjanus

demo.jpg

Figure 15.  LC- janus after Enzyme digestion using EcoR1 and Xhol1.

 7.29 PCR of Standardized fragment

demo.jpg

Figure 16.  We succeeded to get LC-injanus and LC-injanus-m but failed to get LC-sjanus.

enzyme digestion of LC-sjanus-m and LC-injanus-m after transformation

demo.jpg

Figure 17.  Succeeded

8.1 enzyme digestion of LC-sjanus after transformation into PET-28a

demo.jpg

Figure 18.  Failed. The reason may be the ratio of plasmid and LC-sjanus was not suitable. So we considered to change the ratio from 1:5 to 1:10.

8.03 Overlap PCR of LC-sjanus(Standardized fragment)

demo.jpg

Figure 19.  It was clear that we failed.

8.04 enzyme digestion of standardized fragment using Xba1 and Pst1.

demo.jpg

Figure 20.  Enzyme digestion of standardized fragment and PSB1C3.

enzyme digestion after transforming LC-sjanus and LC-injanus into PET-28a.

We tried many times but all failed. We can’t find the reason.

In follow figures, our parts should be about 1200bp length if we had.

8.05

demo.jpg

Figure 21

8.06

demo.jpg

Figure 22

8.08

demo.jpg

Figure 23

8.16 enzyme digestion after transforming LC-sjanus-m into PSB1C3.

demo.jpg

Figure 24. Succeeded. And the after DNA Sequencing results showed it was correct. The standardization of LC-sjanus-m was completed.

enzyme digestion after transforming LC-sjanus into PET-28a.

demo.jpg

Figure 25. Failed

8.18  enzyme digestion after Single colony of LC-sjanus again.

demo.jpg

Figure 26. Failed. Because of lack of fragment and failing many times, we decided to stop any work of LC-sjanus.

8.20 enzyme digestion after transforming LC-injanus and LC-injanus-m into PSB1C3.

demo.jpg

Figure 27. succeeded. But the third lane of LC-injanus may lost the part of injanus. This situation also can be seen in other cloning group. It may due to the mispairing of fragment and plasmid or nonspecific binding when doing PCR.

Summary: We managed to complete the standardization of LC-sjanus-m, LC-injanus and LC-injanus-m except LC-sjanus. And we finally decided to give up LC-sjanus.

Protein expression

LC using N-terminal 6Xhis tag

demo.jpg

demo.jpg

Figure 1 and figure 2.  A is broth before inducing
B is broth after inducing
C is the Precipitation of broken bacteria
D is the liquid flowing through the Ni column
E is medium in the column
F is the MCAC 20 flowing through the Ni column
G is the medium in the column after being washed by MCAC 20
H is the MCAC 30 flowing through the Ni column
I is the medium in the column after being washed by MCAC 30
J is the MCAC 50 flowing through the Ni column
K is the medium in the column after being washed by MCAC 50
L is the MCAC 100 flowing through the Ni column
N is the medium in the column after being washed by MCAC 100
O is the MCAC 200 flowing through the Ni column
P is the medium in the column after being washed by MCAC 200
Q is the MCAC 500 flowing through the Ni column
R is the medium in the column after being washed by MCAC 500
S is the protein sample washed down by MCAC 100
T is the protein sample washed down by MCAC 200

If we successfully expressed LC, it should be about 36KDa. It is clear that LC was not expressed with N-terminal 6xhis tag, so we decided to change expression plasmid which expresses protein with N terminal GST.

demo.jpg

Figure 3.  A is broth before inducing
B is broth after inducing
C is supernatant after centrifugation
D is the Precipitation of broken bacteria
E is the medium in the column after being washed by 1xPBS
F is the medium in the column after being handled by Prescission Protease
G is the protein sample

If we successfully expressed LC, it should be about 60KDa for being fused with GST. And in lane D, we had an obvious stripe in a similar position, which means that LC was all expressed as inclusion body. Due to this, we decided to give up the expression of LC.

LC-sjanus-m using N-terminal 6Xhis tag

demo.jpg

demo.jpg

Figure 1 and figure 2.  A is broth before inducing
B is broth after inducing
C is the Precipitation of broken bacteria
D is the liquid flowing through the Ni column
E is medium in the column
F is the MCAC 20 flowing through the Ni column
G is the medium in the column after being washed by MCAC 20
H is the MCAC 30 flowing through the Ni column
I is the medium in the column after being washed by MCAC 30
J is the MCAC 50 flowing through the Ni column
K is the medium in the column after being washed by MCAC 50
L is the MCAC 100 flowing through the Ni column
N is the medium in the column after being washed by MCAC 100
O is the MCAC 200 flowing through the Ni column
P is the medium in the column after being washed by MCAC 200
Q is the MCAC 500 flowing through the Ni column
R is the medium in the column after being washed by MCAC 500
S is the protein sample washed down by MCAC 100
T is the protein sample washed down by MCAC 200

If we successfully expressed LC-sjanus-m, it should be about 44KDa. It is clear that LC-sjanus-m was not expressed with N-terminal 6xhis tag.

After we failed to express LC with GST, we stopped all expression work about LC. We hope other teams in the future can find way to manage it

Saturday.6.6.2015

PCR on PSB1C3

gene of interstGFP

primer: Xba1-GFPGFP-linker

Xba1-GFP: 5’-GCTCTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTC

GFP-linker: 5’-AGAACCACCACCACCAGAACCACCACCACCTTTGTATAGTTCATC

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

linker- sJanus primer

2μl

sJanus -Pst1 primer

2μl

sJanus-m pET28a(+)

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl


cycle

95

5min

95

30s

150

255

358

460

30s

72

2min

72

10min

4

Forever

 

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

qualitative agarose gel electrophpresisGFP on 55, 58, 60 centigrade worked well.

 

demo.jpg


(The maximum sequence of the maker is 2000bp, and following is 1500, 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 750bp approximately.)

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration: 51.7 ng/μL

Date: Monday,7.13.2015

PCR on pET28a(+)

gene of interstsJanus

primerlinker-sJanussJanus-Pst1

linker-sJanus:5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTAGCAACGGCAACGGCAA

sJanus-Pst1:5’- AACTGCAGCGGCCGCTACTAGTATCACCGACGGCGGTCTG

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

linker- sJanus primer

2μl

sJanus -Pst1 primer

2μl

sJanus pET28a(+)

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

150

2:55℃

3:58℃

460

30s

72

2min

72

10min

4

Forever

 qualitative agarose gel electrophpresissJanus on all centigrades worked

demo.jpg

(The maximum sequence of the maker is 2000bp, and following is 1500, 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 250bp approximately.)

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:70.2ng/μL

Date: Monday,7.13.2015

PCR on pET28a(+)

gene of interstsJanus-m

primerlinker- sJanussJanus-Pst1

linker- sJanus:5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTAGCAACGGCAACGGCAA

SJANUS-Pst1:5’- AACTGCAGCGGCCGCTACTAGTATCACCGACGGCGGTCTG

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

linker- sJanus primer

2μl

sJanus -Pst1 primer

2μl

sJanus-m pET28a(+)

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

150

255

358

460

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresissJanus-m on all centigrades worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 250bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:70.6ng/μL

Date: Monday,7.13.2015

PCR on pET28a(+)

gene of interstinJanus

primerlinker- inJanusinJanus-Pst1

linker- inJanus: 5’- GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTGCACCACT

INJANUS-Pst15’- GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTGCACCACT

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

BFP_F primer

2μl

BFP_R primer

2μl

BFP pSB1C3

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

1:50.1

2:53.4

3:56.4

4:58.5

30s

72

2min

72

10min

4

Forever

 

qualitative agarose gel electrophpresisinJanus didn’t work well. The electrophoretic bands towing phenomenon is obvious.

demo.jpg


(The maximum sequence of the maker is 2000bp, and following is 1500, 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 250bp approximately.)

analyse We suspect is caused by low temperature. So we raise the temperature to conduct the PCR later.

Date: Tuesday,7.14.2015

PCR on pET28a(+)

gene of interstinJanus

primerlinker- inJanusinJanus-Pst1

linker- inJanus:

5’- GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTGCACCACT

inJanus-Pst15’- GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTGCACCACT

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

linker- inJanus

primer

2μl

inJanus

-Pst1 primer

2μl

inJanus

pET28a(+)

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

1:59.4

2:65.4

3:67.0

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisinJanus-m worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 250bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:43.5ng/μL

Date: Tuesday,7.14.2015

PCR on pET28a(+)

gene of interstinJanus-m

primerlinker-inJanus-minJanus-Pst1

linker-inJanus-m:5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTCTACCACTGGC

inJanus

-Pst15’- GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACAGTGCACCACT

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

linker-inJanus-m primer

2μl

inJanus

-Pst1 primer

2μl

inJanus-m pET28a(+)

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

1:59.4

2:65.4

3:67.0

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisinJanus-m on these three centigrades worked

 demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 250bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:55.0ng/μL

Date: Wednesday,7.15.2015

SOE PCR

SOE PCR stands for Splicing by Overlapp Extension PCR. It is a standard overlapp extension procedure, enabling the assembly of genes wihtout performing any cloning, digesting or ligation inbetween. All you need to do ist running PCRs with specific primers. If gene A is the upstream part and gene B has to be assembled downstream of gene A, primer lo of gene A should have an overlapp of around 20 nucleotides complementary to the first 20 nucleotides of gene B. Primer up of gene B should haven a complementary overlapp of 20 nucleotides to the end of gene A.

demo.jpg

gene of interstGFP- sJanus

primerXba1-GFPsJanus-Pst1

temperature: 53, 55, 56, and 58 centigrade.

PCR system100μl

Buffer

10μl

dNTPS

10μl

GFP  fragment

5μl

sJanus fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

Add two kinds of primer (1μl) after 5 cycle.

cycle

95

5min

95

30s

150

256

365

 

 

30s

72

2min

72

10min

95

5min

95

30s

150

256

365

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisGFPsJanus for SOE PCR with PSB1C3 worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:32.2ng/μL

Date: Wednesday,7.15.2015

SOE PCR

gene of interstGFP-sJanus-m

primerXba1-GFPsJanus -Pst1

temperature 53, 55, 56, and 58 centigrade.

 PCR system100μl

Buffer

10μl

dNTPS

10μl

GFP  fragment

5μl

sJanus-m fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

Add two kinds of primer (1μl) after 5 cycle.

cycle

95

5min

95

30s

150

256

365

30s

72

2min

72

10min

95

5min

95

30s

150

256

365

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisGFPsJanus-m for SOE PCR with PSB1C3 worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration: 30.5ng/μL

Date: Wednesday,7.15.2015

SOE PCR

gene of interstGFP- sJanus

primerXba1-GFPinJanus-Pst1

 temperature 55, 56.3 ,57.8 and 58.9 centigrade.

PCR system100μl

Buffer

10μl

dNTPS

10μl

GFP  fragment

5μl

inJanus fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

Add two kinds of primer (1μl) after 5 cycle.

cycle

95

5min

95

30s

155

256.3

357.8

458.9

30s

72

2min

72

10min

95

5min

95

30s

150

256

365

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisGFPinJanus for SOE PCR with PSB1C3 worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:31.1ng/μL

DateWednesday,7.15.2015

SOE PCR

gene of interstGFP-inJanus

primerXba1-GFPinJanus-Pst1

 temperature 55, 56.3 ,57.8 and 58.9 centigrade.

PCR system100μl

Buffer

10μl

dNTPS

10μl

GFP  fragment

5μl

inJanus-m fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

Add two kinds of primer (1μl) after 5 cycle.

cycle

95

5min

95

30s

155

256.3

357.8

458.9

30s

72

2min

72

10min

95

5min

95

30s

150

256

365

30s

72

2min

72

10min

4

Forever

 qualitative agarose gel electrophpresisGFPinJanus-m for SOE PCR with PSB1C3 worked

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:50.3ng/μL

 Date: Thursday,7.16.2015

restriction enzyme digestion

After qualitative agarose gel electrophpresis, all the target sequence are 1000bp approximately.

demo.jpg

Fragment concentration: sJanus is 6.9ng/μL, sJanus-m is 15.3ng/μL, inJanus is 4.6ng/μL, inJanus-m is 7.0ng/μL.

Date: Friday,7.17.2015

We used T4 DNA ligase to connect them at the temperature of 22 centigrade about an hour.

Transformed the expression vectors we had already connected into the Escherichia coli DH5α, then incubated the bacteria for 12h (37).

Date:Saturday,7.18.2015

demo.jpg

Enzymes cut each, but they did not work.

The maximum of marker is 8000bp, and following is 8000, 5000, 3000,1500,1000and 500bp from up and down, our target sequence is about 1000bp approximately. But the bands in this figure are all about 5000bp, so we suspect that the fragments are not connected to plasmid.

AnalyseThe fragments are not connected to plasmid.

Date: Sunday,7.19.2015

We chose another DH5α and trans

formed again.

Date: Monday,7.20.2015

Enzyme cut,

Date: Tuesday,7.21.2015

We design another kind of primer and reverse which contain EcoRI and XhoI site. We have got the standard GFP-sJanus and GFP-sJanus-m fragments, and we got the target fragments used to construct the expression vector by PCR.

gene of interstEcoR1-GFP-sJanus -Xho1EcoR1-GFP-sJanus-m-Xho1

primerEcoR1-GFPsJanus-Xho1

EcoR1-GFP: 5’-CGGAATTCATGCGTAAAGGAGAAGAACTT

sJanus-Xho1: 5’-CCGCTCGAGTCAAGCACCGACGGCC

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

EcoR1-GFP primer

2μl

sJanus-Xho1 primer

2μl

Xba1-GFP-sJanus -Pst1/ Xba1-GFP-sJanus-m-Pst1

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

cycle

95

5min

95

30s

1:55.4

2:56.6

3:57.8

4:60.3

30s

72

2min

72

10min

4

Forever

qualitative agarose gel electrophpresisfragments on these four centigrades all worked well.

demo.jpg

The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately.

Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion.

Fragment concentration:EcoR1-GFP-sJanus -Xho1:55.0ng/μLEcoR1-GFP-sJanus-m-Xho1:60ng/μL

Date: Saturday,8.1.2015

We used enzymes of EcoRI site and XhoI site to digest the PCR product and plasmid. We used T4 DNA ligase to connect them at the temperature of 22 centigrade for about one hour. Then we transformed the expression vectors we had already connected into the Escherichia coli DH5α, then incubated the bacteria for 12h (37)

It has been confirmed that these sequences we get are right when detecting them in company.

Date:Monday,8.10.2015

Transformed the expression vectors(concentration:140.3ng/μL121.4ng/μL) we had already built before (pET28a(+)) into the Escherichia coli BL21, then incubated the bacteria for 12h (37).

1.Get the gene of BFP

1.1 In order to get the fusion protein, we use overlap PCR to construct the fragment(BFP- sJanus & BFP-sJanus-m).We get the gene of BFP by the standard plasmid from iGEM. Then we use PCR to amplify it. The primer we design is as follow:

F:5’- GAATTCGCGGCCGCTTCTAGATGAGCGAACTGATCAAAGAG-3’

(EcoRI and XbaI site are contained).

R:5’- GTGGTGGTGGTCTTGGTGGTGGTGGATTCAGTTTATGACCCAGCTT-3’

(linker-GGGGSGGGGS is contained)

We get the target sequence by PCR at the temperature of 55 ,60 and 65 centigrade.

1.2 detail of the experiment

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

BFP_F primer

2μl

BFP_R primer

2μl

BFP PSB1C3

1μl

Pfu enzyme

0.5μl

ddH2O

34.5μl

 

 

95

5min

95

30s

155

260

365

 

30s

72

2min

72

10min

4

Forever

 

2.Get the gene of BFP- sJanus & BFP-sJanus-m

2.1We get the gene of BFP- sJanus & BFP-sJanus-m by the plasmid pET28a(+) extracting from DH5α. And then we amplify it by PCR. The primer we design is as follow:

F:5’-ACCACCAAGACCACCACCACCAAGAAGCAACGGCAACGGCAAT-3’

(linker-GGGGSGGGGS is contained)

R:5’-CTGCAGGCGGCCGCTACTAGTATCAAGCACCGACGGCGG-3’

( SpeI, NotI, and PstI site are contained)

We get the target sequence by PCR at the temperature of 55, 60 and 65 centigrade.

2.2 Details of experiment

 

 

PCR system:(50μl

Buffer

5μl

dNTPS

5μl

P_F

2μl

P_R

2μl

sJanus PET28a

2μl

Pfu enzyme

0.5μl

ddH2O

33.5μl

 

95

5min

95

30s

155

260

365

 

30s

72

2min

72

10min

4

Forever

 

2.3 Results

demo.jpg

Figure 1 The maximum of marker is 2000bp, and the centigrade of 60 is not suitable for the system because there is a track behind it. Our target is about 250bp approximately

We get the target fragment in the end. All temperature were suitable for the PCR cycle.

3.Overlap PCR

3.1 We use the technique of overlap PCR to get our target sequence. In this experiment, we add BFP and sJanus & sJanus-m which we get in two steps and the BFP’s primer we design and sJanus & sJanus-m’s reverse in the system of PCR. We get the target sequence at the temperature of 55, 60 and 65 centigrade.

3.2 Detail of the experiment

 

PCR system100μl

Buffer

10μl

dNTPS

10μl

BFP    fragment

5μl

sJanus fragment

5μl

Pfu enzyme

1μl

ddH2O

69μl

 

Add two kinds of primer (1μl) after 5 cycle.

 

95

5min

95

30s

150

256

365

 

30s

72

2min

72

10min

95

5min

95

30s

150

256

365

 

30s

72

2min

72

10min

4

Forever

 

 

3.3 Results

demo.jpg


Figure 2

demo.jpg

Figure 3. The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately. We failed at first attempt. But as we change the temperature we use in PCR cycle. We finally get the target fragment.

As we can see from the picture, there are two bands in every channel. One is about 1000bp and another is about 300bp. We do not know the reason of this. But after doing the confirmatory experiment, we are sure the 1000bp band is the target band we want (BFP- sJanus & BFP-sJanus M). We did not get the fragment at first attempt. We change the temperature in PCR cycle.

4. Construct the plasmid

4.1 We design another kind of primer and reverse which contain EcoRI and Hind III site. The condition to amplify it by PCR is same as above all. We choose pET28a(+) as our expression vector. And then, we use enzymes of EcoRI site and XhoI site to digest the PCR product and plasmid. We use T4 DNA ligase to connect them at the temperature of 22 centigrade. We insert the plasmid into competent DH5α. It has been confirmed that these sequences we get are right when detecting them in company.

4.2 detail of the experiment

 

Digest (60μl)    1h

ddH2O

31μl

buffer

6μl

Fd1

1.5μl

Fd2

1.5μl

DNA

20μl

 

 

T4 DNA ligase to connect20μl  1h

Buffer

2μl

T4 DNA ligase

0.5μl

plasmid

X

fragment

5X