Team:Tianjin/GFP

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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

Xba1-GFP primer

2μl

GFP-linker primer

2μl

GFP PSB1C3

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

255

358

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).

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