Team:KIT-Kyoto/Notebook/ATF1/july

ATF1

July 1^st

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 2^nd

We applied DNA sample to the agarose gel electrophoresis.

[Consequence]

The band appeared at the correct size.

 

 

July 4^th

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

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

July5^th・8^th

We purified ATF1.

We digested ATF1 and pET-15b with XhoI.

We purified this ATF1 and pET-15b.

 

 

July 9^th

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 1^st.)

 

 

 

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 12^th

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 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
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 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.