Team:KIT-Kyoto/Notebook/ATF1/september

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Revision as of 06:07, 27 September 2013



ATF1

September 1st

Picked 32 colonies of ATF1 transformants.


September 3rd

Checked the colonies by colony cracking.

No appropriate plasmid DNA was detected.

Digested ATF1 and pET-15b with at 37℃ overnight.

ATF1

24ul

Xho1

1ul

Bpu11021

1ul

BSA

1ul

2NEB Buffer

3ul


pET-15b

33ul

Xho1

1ul

Bpu11021

1ul

BSA

1ul

2NEB Buffer

4ul



September 4th

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

No pET-15b was detected.

Extracted from Blue gel and purified ATF1.



September 11th

PCR reaction was carried out using the primer.

Buffer

50ul

dNTP

20ul

Primer mix

1ul

Genome DNA

0.5ul

KOD-FX

2ul

H2O

26.5ul

Electrophoresed in 1% agarose gel. Verified as ATF1.

Purified PCR products of ATF1.

Digested ATF1 and pET-15b with Xho1 and Bpu11021 at 37℃ for 2 hours.


ATF1

15.5ul

Xho1

1ul

Bpu11021

1ul

BSA

0.5ul

2NEB Buffer

2ul

Added 1ul of BAP to the solution of pET-15b and incubated it at 37℃ for 30 minutes.

Ligated of pET-15b with ATF1 and transformed.

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

Isolated and purified them.



September 12th

Picked 32 colonies of ATF1 transformants.



September 13th

Checked the colonies by colony cracking.

Picked up the appropriate colonies and cultured in the LB in 3ml ampicillin(+) medium at 37℃ for 5 hours.

Miniprepped plasmid DNA.

Digested plasmid DNA with Xho1 and Bpu11021 at 37℃ for 2 hours.

ATF1 into pET-15b

15.5ul

Xho1

1ul

Bpu11021

1ul

BSA

0.5ul

2NEB Buffer

2ul

Applied pET-15b to the agarose gel electrophoresis.

No band was detected.

Plasmid DNA was digested with Xho1 and Bpu11021 at 37℃ (overnight).


ATF1 into pET-15b

15.5ul

Xho1

1ul

Bpu11021

1ul

BSA

0.5ul

2NEB Buffer

2ul




September 14th

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

No band was detected.

Digested ATF1 and pET-15b with Bpu11021 at 37℃ overnight.


ATF1

26ul

Bpu11021

1ul

Bpu Buffer

3ul


pET-15b

26ul

Bpu11021

1ul

Bpu Buffer

3ul



September 15th

Purified ATF1 and pET-15b.

Digested ATF1 and pET-15b with Xho1.

ATF1

26ul

Xho1

1ul

Bpu Buffer

3ul


pET-15b

26ul

Xho11021

1ul

Bpu Buffer

3ul


pET-15b and ATF1 were applied to electrophoresis to the Blue gel and purified.

Ligated ATF1 with pET-15b and transformed them into E. coli cells.



September 16th

Picked 32 colonies of ATF1 transformants.



September 18th

Checked the colonies by colony cracking.

Picked up the colonies and cultured in 3ml LB medium with ampicillin at 37℃ (overnight).



September 19th

plasmid DNA was purified and digested with Xho1 and Bpu11021 at 37℃ for 2 hours.

ATF1 into pET-15b

15.5ul

Xho1

1ul

Bpu11021

1ul

BSA

0.5ul

2NEB Buffer

2ul

ATF1 and pET-15b were applied to electrophoresis to the agarose gel.

No band was detected.

Digested ATF1 and pET-15b with Xho1 and Blp1.

ATF1

15.5ul

Xho1

1ul

Blp1

1ul

BSA

0.5ul

Buffer

2ul


pET-15b

15.5ul

Xho1

1ul

Blp1

1ul

BSA

0.5ul

Buffer

2ul


Added 1ul of BAP to the solution of pET-15b and incubated it at 37℃ for 30 minutes.

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

Isolated and purified them.

The ATF1 was ligated with pET-15b, and transformed into E. coli cells.



September 20th

Picked 64 colonies of ATF1 transformants.





September 21st

Checked the colonies by colony cracking.

Picked up colonies and cultured in the LB in 3ml LB medium with ampicillin at 37℃ (overnight).


September 22nd

Miniprepped plasmid DNA and digested it with Xho1 and Blp1 at 37℃ for 2 hours.

Applied ATF1 into pET-15b to the agarose gel electrophoresis.

No band was detected.