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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 XhoⅠat 37℃ for 90 minutes.
|
pET-15b |
88ul |
|
XhoⅠ Buffer |
10ul |
|
XhoⅠ |
2ul |
|
Total |
100ul |
Purified pET-15b DNA was dissolved in 34ul of H2O.
Digested pET-15b with Bpu1102Ⅰ at 37℃ for 90 minutes.
|
pET-15b |
34ul |
|
Bpu1102Ⅰ Buffer |
4ul |
|
Bpu1102Ⅰ |
2ul |
|
Total |
40ul |
Added 1uL of BAP to the solution and incubated it at 37℃ for 30 minutes.
Applied ATF1 and pET-15b to the blue gel electrophoresis.
Results: No band was detected.
Cultivated pET-15b in ampicillin (+) liquid LB overnight.
August 7th
Minipreped pET-15b DNA.
Digested pET-15b with XhoⅠ at 37℃ for 3 hours.
|
pET-15b |
88ul |
|
XhoⅠ Buffer |
10ul |
|
XhoⅠ |
2ul |
|
Total |
100ul |
Purified it and dissolved in 26ul of H2O.
Digested pET-15b with Bpu1102Ⅰ at 37℃ for 3 hours.
|
pET-15b |
26ul |
|
Bpu1102Ⅰ Buffer |
3ul |
|
Bpu1102Ⅰ |
1ul |
|
Total |
30ul |
Added 1uL of BAP to this solution and incubated it at 37℃ for 30 minutes.
Applied ATF1 and pET-15b to the blue gel electrophoresis.
August 8th
Digested pET-15b with XhoⅠ at 37℃ overnight.
|
pET-15b |
89ul |
|
XhoⅠ Buffer |
10ul |
|
XhoⅠ |
1ul |
|
Total |
100ul |
August 9th
Purified pET-15b (prepared on 8/8) and added 26ul of H2O.
Digested pET-15b with Bpu1102Ⅰ at 37℃ for 3 hours.
|
pET-15b |
26ul |
|
Bpu1102Ⅰ Buffer |
3ul |
|
Bpu1102Ⅰ |
1ul |
|
Total |
30ul |
Added 1uL of BAP to this solution and incubate it at 37℃ for 30 minutes.
Applied pET-15b to the blue gel electrophoresis.
Isolated and Purified it.
Ligation of pET-15b with ATF1 (prepared 7/19,8/6) at room temperature for 15 minutes.
|
ATF1 |
5ul |
|
pET-15b |
5ul |
|
Ligation MIX |
10ul |
|
Total |
20ul |
The ATF1 into pET-15b was transformed into E.coli cells.
Cultivated transformants on LB ampicillin (+) plate at37℃ 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℃ for 4 hours.
Miniprepped plasmid DNA.
August 13th
Digested plasmid DNA (prepared on 8/12) with HindⅢ at 37℃ for 90 minutes.
|
Plasmid DNA |
5ul |
|
Buffer |
2ul |
|
H2O |
12ul |
|
HindⅢ |
1ul |
|
Total |
20ul |
Applied pET-15b to the agarose gel electrophoresis.
Digested plasmid DNA (prepared on 8/12) with HindⅢ at 37℃ for 90 minutes.
|
Plasmid DNA |
10ul |
|
Buffer |
2ul |
|
H2O |
7.25ul |
|
HindⅢ |
0.75ul |
|
Total |
20ul |
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℃.
August 26th
We performed PCR to amplify the ATF1 gene.
|
Buffer |
50ul |
|
dNTP |
20ul |
|
Primer mix |
1ul |
|
Genome DNA |
0.5ul |
|
KOD-FX |
2ul |
|
H2O |
26.5ul |
August 27th
Purified PCR products (prepared on 8/26) and added 30ul of H2O.
Digested ATF1 with HindⅢ.
|
ATF1l |
5ul |
|
Buffer |
2ul |
|
HindⅢ |
2ul |
|
H2O |
11ul |
Digested pET-15b and ATF1 with XhoⅠ.
|
ATF1 |
25ul |
|
Buffer |
3ul |
|
XhoⅠ |
2ul |
|
pET-15b |
100ul |
|
Buffer |
11.3ul |
|
XhoⅠ |
2ul |
Purified it and added 25ul of H2O.
Digested pET-15b and ATF1 with Bpu1102Ⅰ.
|
ATF1 |
25ul |
|
Buffer |
3ul |
|
Bpu1102Ⅰ |
2ul |
|
pET-15b |
25ul |
|
Buffer |
3ul |
|
Bpu1102Ⅰ |
2ul |
Applied pET-15b and ATF1 to the blue gel electrophoresis.
Purified PCR products.
August 28th
Digested ATF1 (prepared on 8/27) with EcoRⅠ .
|
ATF1l |
5ul |
|
Buffer |
1ul |
|
EcoRⅠ |
2ul |
|
H2O |
2ul |
Applied it to the agarose gel electrophoresis.
Digested ATF1 (prepared on 8/27) and pET-15b with XhoⅠ.
|
ATF1 |
25ul |
|
Buffer |
3ul |
|
XhoⅠ |
2ul |
|
pET-15b |
100ul |
|
Buffer |
11.3ul |
|
XhoⅠ |
2ul |
August 29th
Applied ATF1 and pET-15b to the blue gel electrophoresis.
Ligated it.
|
ATF1 |
10ul |
|
pET-15b |
10ul |
|
Ligation MIX |
10ul |
Carried out transformation.
August 30th
Checked the colonies by colony cracking.
We performed PCR to amplify the ATF1 gene.
|
Buffer |
50ul |
|
dNTP |
20ul |
|
Primer mix |
1ul |
|
Genome DNA |
0.5ul |
|
KOD-FX |
2ul |
|
H2O |
26.5ul |
August 31st
Purified PCR products and dissolved 30ul of H2O.
Purified pET-15b and added 33ul of H2O.
Digested ATF1 and pET-15b with XhoⅠ and Bpu1102Ⅰ.
|
NEB Buffer |
3ul |
|
ATF1 |
24ul |
|
BSA |
1ul |
|
XhoⅠ |
1ul |
|
Bpu1102Ⅰ |
1ul |
|
NEB2 Buffer |
4ul |
|
pET-15b |
33ul |
|
BSA |
1ul |
|
XhoⅠ |
1ul |
|
Bpu1102Ⅰ |
1ul |
Added 1uL of BAP to pET-15b and incubated them at 37℃ for 30 minutes.
Applied pET-15b to the blue gel electrophoresis.
Isolated and purified it.
Ligation of pET-15b with ATF1 (prep).
|
ATF1 |
10ul |
|
pET-15b |
10ul |
|
Ligation MIX |
10ul |
Transformed ATF1 into pET-15b E. coli cells.
