Team:KIT-Kyoto/Notebook/ATF1/september

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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 XhoI and Bpu1102I at 37˚C overnight.

ATF1

24µL

XhoI

1µL

Bpu1102I

1µL

BSA

1µL

NEB Buffer 2

3µL

 

pET-15b

33µL

XhoI

1µL

Bpu1102I

1µL

BSA

1µL

NEB Buffer 2

4µL

 

 

September 4th       

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

No pET-15b was detected.

ATF1 was extracted from the blue gel.

 

 

September 11th

PCR reaction was carried out using the primer.

(Forward : CGATCCTCGAGTGGCCTGGTATTGTCATCGCGTTG

Reverse : CGATCGCTCAGCAACCAACCAAAGCCGAGGGAGTG)

Buffer

50µL

dNTP

20µL

Primer mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

Electrophoresed in 1% agarose gel. Verified as ATF1.

Purified the PCR products of ATF1.

Digested ATF1 and pET-15b with XhoI and Bpu1102I at 37˚C for 2 hours.

 

ATF1

15.5µL

XhoI

1µL

Bpu1102I

1µL

BSA

0.5µL

NEB Buffer 2

2µL

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

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

Isolated and purified them.

Ligated pET-15b and ATF1 and transformed.

 

 

September 12th

Picked 32 colonies of ATF1 transformants.

 

 

September 13th

Checked the colonies by colony cracking.

Picked up the appropriate colonies and cultured in 3mL LB medium with ampicillin at 37˚C for 5 hours.

Miniprepped plasmid DNA.

Digested plasmid DNA with XhoI and Bpu1102I at 37˚C for 2 hours.

ATF1 into pET-15b

15.5µL

XhoI

1µL

Bpu1102I

1µL

BSA

0.5µL

NEB Buffer 2

2µL

Applied pET-15b to the agarose gel electrophoresis.

No band was detected.

Plasmid DNA was digested with XhoI and Bpu1102I at 37˚C (overnight).

 

ATF1 into pET-15b

15.5µL

XhoI

1µL

Bpu1102I

1µL

BSA

0.5µL

NEB Buffer 2

2µL

 

 

 

September 14th

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

No band was detected.

Digested ATF1 and pET-15b with Bpu1102I at 37˚C overnight.

 

ATF1

26µL

Bpu1102I

1µL

Bpu1102I Buffer

3µL

 

pET-15b

26µL

Bpu1102I

1µL

Bpu1102I Buffer

3µL

 

 

September 15th

Purified ATF1 and pET-15b.

Digested ATF1 and pET-15b with XhoI.

ATF1

26µL

XhoI

1µL

Bpu1102I Buffer

3µL

 

pET-15b

26µL

XhoI

1µL

Bpu1102I Buffer

3µL

 

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

Ligated ATF1 and 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˚C (overnight).

 

 

September 19th

plasmid DNA was purified and digested with XhoI and Bpu1102I at 37˚C for 2 hours.

ATF1 into pET-15b

15.5µL

XhoI

1µL

Bpu1102I

1µL

BSA

0.5µL

NEB Buffer 2

2µL

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

No band was detected.

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

ATF1

15.5µL

XhoI

1µL

BlpI

1µL

BSA

0.5µL

Buffer

2µL

 

pET-15b

15.5µL

XhoI

1µL

BlpI

1µL

BSA

0.5µL

Buffer

2µL

 

Added 1µL of BAP to the solution of pET-15b and incubated it at 37˚C 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˚C (overnight).

 

September 22nd

Miniprepped plasmid DNA and digested it with XhoI and BlpI at 37˚C for 2 hours.

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

No band was detected.