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Team:KIT-Kyoto/Notebook/ATF1/july
From 2013.igem.org
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 |
July5th・8th
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.
