Team:Tianjin/GFP

内页

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

 


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