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Team:KIT-Kyoto/Notebook/ATF1/august
From 2013.igem.org
ATF1
August 6th
The ATF1 gene was purified (prepared on 7/19)
Digested pET-15b with XhoI at 37 ˚C for 90 minutes.
|
pET-15b |
88µL |
|
XhoI Buffer |
10µL |
|
XhoI |
2µL |
|
Total |
100µL |
Purified pET-15b DNA was dissolved in 34µL of H2O.
Digested pET-15b with Bpu1102I at 37 ˚C for 90 minutes.
|
pET-15b |
34µL |
|
Bpu1102I Buffer |
4µL |
|
Bpu1102I |
2µL |
|
Total |
40µL |
Added 1uL of BAP to the solution and incubated it at 37 ˚C for 30 minutes.
Applied ATF1 and pET-15b to the blue gel electrophoresis.
Results: No band was detected.
Cultivated pET-15b in 3mL LB medium with ampicillin overnight.
August 7th
Minipreped pET-15b DNA.
Digested pET-15b with XhoI at 37˚C for 3 hours.
|
pET-15b |
88µL |
|
XhoI Buffer |
10µL |
|
XhoI |
2µL |
|
Total |
100µL |
Purified it and dissolved in 26µL of H2O.
Digested pET-15b with Bpu1102I at 37˚C for 3 hours.
|
pET-15b |
26µL |
|
Bpu1102I Buffer |
3µL |
|
Bpu1102I |
1µL |
|
Total |
30µL |
Added 1µL of BAP to this solution and incubated it at 37˚C for 30 minutes.
Applied ATF1 and pET-15b to the blue gel electrophoresis.
August 8th
Digested pET-15b with XhoI at 37˚C overnight.
|
pET-15b |
89µL |
|
XhoI Buffer |
10µL |
|
XhoI |
1µL |
|
Total |
100µL |
August 9th
Purified pET-15b (prepared on 8/8) and added 26ul of H2O.
Digested pET-15b with Bpu1102I at 37˚C for 3 hours.
|
pET-15b |
26 |
|
Bpu1102I Buffer |
3µL |
|
Bpu1102I |
1µL |
|
Total |
30µL |
Added 1µL of BAP to this solution and incubate it at 37˚C for 30 minutes.
Applied pET-15b to the blue gel electrophoresis.
Isolated and Purified it.
Ligation pET-15b and ATF1 (prepared 7/19,8/6) at room temperature for 15 minutes.
|
ATF1 |
5µL |
|
pET-15b |
5µL |
|
Ligation MIX |
10µL |
|
Total |
20µL |
The ATF1 into pET-15b was transformed into E.coli cells.
Cultivated transformants on LB plate with ampicillin at37˚C overnight.
August 10th
Picked 48 colonies of ATF1 transformants.
August 12th
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in the LB in 3mL ampicillin(+) medium at 37˚C for 4 hours.
Miniprepped plasmid DNA.
August 13th
Digested plasmid DNA (prepared on 8/12) with HindIII at 37˚C for 90 minutes.
|
Plasmid DNA |
5µL |
|
Buffer |
2µL |
|
H2O |
12µL |
|
HindIII |
1µL |
|
Total |
20µL |
Applied pET-15b to the agarose gel electrophoresis.
Digested plasmid DNA (prepared on 8/12) with HindIII at 37˚C for 90 minutes.
|
Plasmid DNA |
10µL |
|
Buffer |
2µL |
|
H2O |
7.25µL |
|
HindIII |
0.75µL |
|
Total |
20µL |
Applied pET-15b to the agarose gel electrophoresis.
Again, picked up the appropriate colonies and cultured in the 3mL LB medium with ampicillin at 37˚C.
August 26th
We performed PCR to amplify the ATF1 gene.
|
Buffer |
50µL |
|
dNTP |
20µL |
|
Primer mix |
1µL |
|
Genome DNA |
0.5µL |
|
KOD-FX |
2µL |
|
H2O |
26.5µL |
August 27th
Purified PCR products (prepared on 8/26) and added 30µL of H2O.
Digested ATF1 with HindIII.
|
ATF1 |
5µL |
|
Buffer |
2µL |
|
HindIII |
2µL |
|
H2O |
11µL |
Digested pET-15b and ATF1 with XhoI.
|
ATF1 |
25µL |
|
Buffer |
3µL |
|
XhoI |
2µL |
|
pET-15b |
100µL |
|
Buffer |
11.3µL |
|
XhoI |
2µL |
Purified it and added 25µL of H2O.
Digested pET-15b and ATF1 with Bpu1102I.
|
ATF1 |
25µL |
|
Buffer |
3µL |
|
Bpu1102I |
2µL |
|
pET-15b |
25µL |
|
Buffer |
3µL |
|
Bpu1102I |
2µL |
Applied pET-15b and ATF1 to the blue gel electrophoresis.
Purified PCR products.
August 28th
Digested ATF1 (prepared on 8/27) with EcoRI.
|
ATF1 |
5µL |
|
Buffer |
1µL |
|
EcoRI |
2µL |
|
H2O |
2µL |
Applied it to the agarose gel electrophoresis.
Digested ATF1 (prepared on 8/27) and pET-15b with XhoI.
|
ATF1 |
25µL |
|
Buffer |
3µL |
|
XhoI |
2µL |
|
pET-15b |
100µL |
|
Buffer |
11.3µL |
|
XhoI |
2µL |
August 29th
Applied ATF1 and pET-15b to the blue gel electrophoresis.
Ligated it.
|
ATF1 |
10µL |
|
pET-15b |
10µL |
|
Ligation MIX |
10µL |
Carried out transformation.
August 30th
Checked the colonies by colony cracking.
We performed PCR to amplify the ATF1 gene.
|
Buffer |
50µL |
|
dNTP |
20µL |
|
Primer mix |
1µL |
|
Genome DNA |
0.5µL |
|
KOD-FX |
2µL |
|
H2O |
26.5µL |
August 31st
Purified PCR products and dissolved 30µL of H2O.
Purified pET-15b and added 33µL of H2O.
Digested ATF1 and pET-15b with XhoI and Bpu1102I.
|
NEB Buffer 2 |
3µL |
|
ATF1 |
24µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
Bpu1102I |
1µL |
|
NEB Buffer 2 |
4µL |
|
pET-15b |
33µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
Bpu1102I |
1µL |
Added 1µL of BAP to pET-15b and incubated them at 37˚C for 30 minutes.
Applied pET-15b to the blue gel electrophoresis.
Isolated and purified it.
Ligation of pET-15b with ATF1 (prep).
|
ATF1 |
10µL |
|
pET-15b |
10µL |
|
Ligation MIX |
10µL |
Transformed ATF1 into pET-15b E. coli cells.
