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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 |
July5th・8th
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.
