Team:KIT-Kyoto/Notebook/ATF1/august

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ATF1

August 6th

The ATF1 gene was purified (prepared on 7/19)

Digested pET-15b with XhoⅠat 37℃ for 90 minutes.

pET-15b

88ul

XhoⅠ Buffer

10ul

XhoⅠ

2ul

Total

100ul

Purified pET-15b DNA was dissolved in 34ul of H2O.

Digested pET-15b with Bpu1102Ⅰ at 37℃ for 90 minutes.

pET-15b

34ul

Bpu1102Ⅰ Buffer

4ul

Bpu1102Ⅰ

2ul

Total

40ul

Added 1uL of BAP to the solution and incubated it at 37℃ for 30 minutes.

Applied ATF1 and pET-15b to the blue gel electrophoresis.

Results: No band was detected.

Cultivated pET-15b in ampicillin (+) liquid LB overnight.


August 7th

Minipreped pET-15b DNA.

Digested pET-15b with XhoⅠ at 37℃ for 3 hours.

pET-15b

88ul

XhoⅠ Buffer

10ul

XhoⅠ

2ul

Total

100ul

Purified it and dissolved in 26ul of H2O.

Digested pET-15b with Bpu1102Ⅰ at 37℃ for 3 hours.

pET-15b

26ul

Bpu1102Ⅰ Buffer

3ul

Bpu1102Ⅰ

1ul

Total

30ul

Added 1uL of BAP to this solution and incubated it at 37℃ for 30 minutes.

Applied ATF1 and pET-15b to the blue gel electrophoresis.


August 8th

Digested pET-15b with XhoⅠ at 37℃ overnight.

pET-15b

89ul

XhoⅠ Buffer

10ul

XhoⅠ

1ul

Total

100ul



August 9th

Purified pET-15b (prepared on 8/8) and added 26ul of H2O.

Digested pET-15b with Bpu1102Ⅰ at 37℃ for 3 hours.

pET-15b

26ul

Bpu1102Ⅰ Buffer

3ul

Bpu1102Ⅰ

1ul

Total

30ul

Added 1uL of BAP to this solution and incubate it at 37℃ for 30 minutes.

Applied pET-15b to the blue gel electrophoresis.

Isolated and Purified it.

Ligation of pET-15b with ATF1 (prepared 7/19,8/6) at room temperature for 15 minutes.

ATF1

5ul

pET-15b

5ul

Ligation MIX

10ul

Total

20ul

The ATF1 into pET-15b was transformed into E.coli cells.

Cultivated transformants on LB ampicillin (+) plate at37℃ overnight.


August 10th

Picked 48 colonies of ATF1 transformants.



August 12th

Checked the colonies by colony cracking.

Picked up the appropriate colonies and cultured in the LB in 3ml ampicillin(+) medium at 37℃ for 4 hours.

Miniprepped plasmid DNA.



August 13th

Digested plasmid DNA (prepared on 8/12) with HindⅢ at 37℃ for 90 minutes.

Plasmid DNA

5ul

Buffer

2ul

H2O

12ul

HindⅢ

1ul

Total

20ul

Applied pET-15b to the agarose gel electrophoresis.

Digested plasmid DNA (prepared on 8/12) with HindⅢ at 37℃ for 90 minutes.

Plasmid DNA

10ul

Buffer

2ul

H2O

7.25ul

HindⅢ

0.75ul

Total

20ul

Applied pET-15b to the agarose gel electrophoresis.

Again, picked up the appropriate colonies and cultured in the 3ml LB medium with ampicillin at 37℃.




August 26th

We performed PCR to amplify the ATF1 gene.

Buffer

50ul

dNTP

20ul

Primer mix

1ul

Genome DNA

0.5ul

KOD-FX

2ul

H2O

26.5ul



August 27th

Purified PCR products (prepared on 8/26) and added 30ul of H2O.

Digested ATF1 with HindⅢ.

ATF1l

5ul

Buffer

2ul

HindⅢ

2ul

H2O

11ul

Digested pET-15b and ATF1 with XhoⅠ.

ATF1

25ul

Buffer

3ul

XhoⅠ

2ul


pET-15b

100ul

Buffer

11.3ul

XhoⅠ

2ul

Purified it and added 25ul of H2O.

Digested pET-15b and ATF1 with Bpu1102Ⅰ.

ATF1

25ul

Buffer

3ul

Bpu1102Ⅰ

2ul


pET-15b

25ul

Buffer

3ul

Bpu1102Ⅰ

2ul

Applied pET-15b and ATF1 to the blue gel electrophoresis.

Purified PCR products.



August 28th

Digested ATF1 (prepared on 8/27) with EcoRⅠ .

ATF1l

5ul

Buffer

1ul

EcoRⅠ

2ul

H2O

2ul

Applied it to the agarose gel electrophoresis.

Digested ATF1 (prepared on 8/27) and pET-15b with XhoⅠ.

ATF1

25ul

Buffer

3ul

XhoⅠ

2ul


pET-15b

100ul

Buffer

11.3ul

XhoⅠ

2ul



August 29th

Applied ATF1 and pET-15b to the blue gel electrophoresis.

Ligated it.

ATF1

10ul

pET-15b

10ul

Ligation MIX

10ul

Carried out transformation.



August 30th

Checked the colonies by colony cracking.

We performed PCR to amplify the ATF1 gene.

Buffer

50ul

dNTP

20ul

Primer mix

1ul

Genome DNA

0.5ul

KOD-FX

2ul

H2O

26.5ul




August 31st

Purified PCR products and dissolved 30ul of H2O.

Purified pET-15b and added 33ul of H2O.

Digested ATF1 and pET-15b with XhoⅠ and Bpu1102Ⅰ.

NEB Buffer

3ul

ATF1

24ul

BSA

1ul

XhoⅠ

1ul

Bpu1102Ⅰ

1ul


NEB2 Buffer

4ul

pET-15b

33ul

BSA

1ul

XhoⅠ

1ul

Bpu1102Ⅰ

1ul

Added 1uL of BAP to pET-15b and incubated them at 37℃ for 30 minutes.

Applied pET-15b to the blue gel electrophoresis.

Isolated and purified it.

Ligation of pET-15b with ATF1 (prep).

ATF1

10ul

pET-15b

10ul

Ligation MIX

10ul

Transformed ATF1 into pET-15b E. coli cells.