Team:KIT-Kyoto/Notebook/ATF1/july

ATF1

July 1^st

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 2^nd

We applied DNA sample to the agarose gel electrophoresis.

[Consequence]

The band appear the correct place.

July 4^th

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

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

July5^th・8^th

We purified ATF1.

We digested ATF1 and pET-15b with Xho1.

We purified this ATF1 and pET-15b.

July 9^th

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 1^st

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 12^th

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 17^th

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 18^th

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 19^th

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.