Team:KIT-Kyoto/Notebook/ATF1/july

From 2013.igem.org




ATF1

July 1st

We performed PCR at 53˚C and 54˚C. (annealing temp.)

We applied DNA sample to the agarose gel electrophoresis.

-53˚C ver.-

We detected several bands.

-54˚C ver.-

The bands seems wrong size.

1. We applied DNA sample to the blue gel electrophoresis and purified DNA.

 [Consequence]

The band appeared 1600bp.

2. We performed PCR at 51˚C and 52˚C. (annealing temp.)

Buffer

50µL

dNTP

20µL

primer mix

1µL

PCR product

4µL

KOD-FX

2µL

H2O

23µL

 

 

July 2nd

We applied DNA sample to the agarose gel electrophoresis.

[Consequence]

The band appeared at the correct size.

 

 

July 4th

We purified DNA sample (PCR product on July 1st.)

We digested ATF1 with HindIII.

We applied DNA sample to the agarose gel electrophoresis.

[Consequence]

The band appeared at the correct size.

We performed PCR for ATF1.

Buffer

50µL

dNTP

20µL

primer mix

1µL

PCR product

4µL

KOD-FX

2µL

H2O

23µL

July5th8th

We purified ATF1.

We digested ATF1 and pET-15b with XhoI.

We purified this ATF1 and pET-15b.

 

 

July 9th

We digested ATF1 and pET-15b with Bpu1102I.

ATF1

25µL

Buffer

3µL

Bpu1102I

2µL

total

30µL

 

pET-15b

25µL

Buffer

3µL

Bpu1102I

2µL

total

30µL

We incubated at 37˚C(30min)

We added 1uL BAP to vector.

We incubated at 37˚C(30min)

We applied DNA sample to the blue gel electrophoresis.

[Consequence]

The band did not appear.

We performed PCR DNA sample (PCR product on July 1st.)

 

 


 

July 11th

We purified ATF1

We digested ATF1 with XhoI.

ATF1

25µL

XhoI

2µL

Buffer(10xH)

3µL

Total

30µL

 

pET-15b

88µL

XhoI

2µL

Buffer(10xH)

10µL

total

100µL

 

We applied DNA sample to the agarose gel electrophoresis.

We purified DNA sample.

We digested ATF1 and Vector with Bpu1102I.

ATF1

25µL

Buffer

3µL

Bpu1102I

2µL

total

30µL

 

pET-15b

34µL

Buffer

2µL

Bpu1102I

4µL

total

40µL

We applied DNA sample to the blue gel electrophoresis.

[result]

Failed

 

 

 

 

 

 

 

 

 

 

July 12th

We performed PCR at 51˚C and 48˚C

Buffer

50µL

dNTP

20µL

Primer mix

1µL

genome

0.5µL

KOD-FX

2µL

H2O

26.5µL

We applied DNA sample to the blue gel electrophoresis.

[result] Failed

We performed PCR at 51˚C

Buffer

50µL

dNTP

20µL

Primer mix

1µL

ATF1plasmid

0.5µL

KOD-FX

2µL

H2O

26.5µL

 

 

July 17th

We applied DNA sample to the agarose gel electrophoresis.

We performed PCR

Buffer

50µL

dNTP

20µL

Primer mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

We extracted sample DNA for gel

We applied DNA sample to the agarose gel electrophoresis.

We performed PCR.

Buffer

50µL

dNTP

20µL

Primer mix

1µL

Genome DNA

0.5µL

KOD-FX

4µL

H2O

25µL

 

 

July 18th

We extracted sample DNA for gel

We performed PCR.

Buffer

50µL

dNTP

20µL

Primer mix

1µL

ATF1

2µL

KOD-FX

2µL

H2O

25µL

We applied DNA sample to the agarose gel electrophoresis.

We performed PCR to use KOD-FXNeo and KOD-FX

We applied DNA sample to the agarose gel electrophoresis.

 

 

 


 

July 19th

We purified PCR products.

We digested sample DNA with HindIII.

We digested pet-15b and ATF1 with XhoI.

ATF1

25µL

XhoI

2µL

Buffer(10xH)

3µL

total

30µL

 

pET-15b

34µL

XhoI

2µL

Buffer(10xH)

4µL

total

40µL

We purified them.

We digested pET-15b and ATF1 with Bpu1102I.

pET-15b

25µL

Buffer

3µL

Bpu1102I

2µL

total

30µL

              

ATF1

25µL

Buffer

3µL

Bpu1102I

2µL

total

30µL

We applied DNA sample to the blue gel electrophoresis.