Team:KIT-Kyoto/Notebook/ATF1/july

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Revision as of 05:58, 27 September 2013



ATF1

July 1st

We performed PCR at 53℃ and 54℃.

We applied DNA sample to the agarose gel electrophoresis.

-53℃ ver-

We detected several bands.

-54℃ 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℃ and 52℃.

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 appear the correct place.



July 4th

We purified DNA sample that PCR product in July 1st.

We digested ATF1 with HindⅢ.

We applied DNA sample to the agarose gel electrophoresis.

[Consequence]

The band appear correct place.

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 Xho1.

We purified this ATF1 and pET-15b.



July 9th

We digested ATF1 and pET-15b with Bpu11021I.

ATF1

25μl

Buffer

3μl

Bpu1102 I

2μl

total

30μl


pET-15b

25μl

Buffer

3μl

Bpu1102 I

2μl

total

30μl

We incubated at 37℃(30min)

We add 1uL BAP to

We incubated at 37℃(30min)

We applied DNA sample to the blue gel electrophoresis.

[Consequence]

The band did not appear.

We performed PCR DNA sample that PCR product in July 1st




July 11th

We purified ATF1

We digested ATF1 with Xho1.

ATF1

25μl

Xho1

2μl

Buffer(10×H)

3μl

total

30μl


pET-15b

88μl

Xho1

2μl

Buffer(10×H)

10μl

total

100μl


We applied DNA sample to the agarose gel electrophoresis.

We purified DNA sample

We digested ATF1 and Vector with Bpu11021.

ATF1

25μl

Buffer

3μl

Bpu1102 I

2μl

total

30μl


pET-15b

34μl

Buffer

2μl

Bpu1102 I

4μl

total

40μl

We applied DNA sample to the blue gel electrophoresis.

[result]

Failed











July 12th

We performed PCR at 51℃ and 48℃

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℃

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

TD>

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 Xho1.

ATF1

25μl

Xho1

2μl

Buffer(10×H)

3μl

total

30μl


pET-15b

34μl

Xho1

2μl

Buffer(10×H)

4μl

total

40μl

We purified them.

We digested pET-15b and ATF1 with Bpu1102Ⅰ.

pET-15b

25μl

Buffer

3μl

Bpu1102 I

2μl

total

30μl

ATF1

25μl

Buffer

3μl

Bpu1102 I

2μl

total

30μl

We applied DNA sample to the blue gel electrophoresis.