Team:KIT-Kyoto/Notebook/ATF1/june

From 2013.igem.org




ATF1

June 4th

Minipreped ATF1(prepared on 5/31).

 

 

June 6th

Performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

Forward: TACGCCTCGAGATGAATGAAATCGATGAGAAAAATCAGGCCCC

Reverse: ATATTGCTTAGCAGGGCCTAAAAGGAGAGCTTTGTAAATGGAGCAAAGC

35cycle

 

 

June 7th

Electrophoresed PCR products in 1% agarose gel. No DNA was detected.

 

 

June 10th

Performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

Predenature 94˚C 2min

Denature 98˚C 10sec

Annealing 51˚C 30sec

Extension 68˚C 1min30sec

(30 cycles)

 

 

 

 

June 11th

Electrophoresed PCR products in 1% agarose gel.

We detected the correct size of DNA band.

We performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

ATF1(PCR product)

0.5µL

KOD-FX

2µL

H2O

26.5µL

Predenature 94˚C 2min

Denature 98˚C 10sec

Annealing 51˚C 30sec

Extension 68˚C 1min30sec

(30 cycles)

 

 

June 12th

Electrophoresed PCR products in 1% agarose gel.

We detected the correct size of DNA band.

 

 

June 13th

Made competent cells.

 

 

June 17th

Purified the PCR product.

 

 

June 20th

Electrophoresed ATF1(prepared on 6/17) in 1% agarose gel.

Many DNA fragments were detected (failed PCR).

 

 

 

 

 

June 21st

PCR was carried out.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

ATF1(PCR product)

0.5µL

KOD-FX

2µL

H2O

26.5µL

30cycle (Annealing:52˚C)

PCR products were electrophoresed in 1% agarose gel.

Many DNA fragments were detected (failed PCR).

PCR was carried out.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

ATF1(PCR product)

0.5µL

KOD-FX

2µL

H2O

26.5µL

30cycle (Annealing:53˚C)

 

 

June 24th

PCR products were electrophoresed in 1% agarose gel.

Isolated and purified ATF1 from 1% agarose gel.

We performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

ATF1(PCR product)

0.5µL

KOD-FX

2µL

H2O

26.5µL

30cycle (Annealing:52˚C)

 

 

 

 

 

 

June 27th

PCR products were electrophoresed in 1% agarose gel.

Four DNA fragments were detected

 

 

June 28th

We performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer Mix

1µL

ATF1(PCR product)

0.5µL

KOD-FX

2µL

H2O

26.5µL

30cycle (Annealing:53˚Cand54˚C)