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Team:KIT-Kyoto/Notebook/ATF1/september
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Revision as of 06:07, 27 September 2013
ATF1
September 1st
Picked 32 colonies of ATF1 transformants.
September 3rd
Checked the colonies by colony cracking.
No appropriate plasmid DNA was detected.
Digested ATF1 and pET-15b with at 37℃ overnight.
|
ATF1 |
24ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
1ul |
|
2NEB Buffer |
3ul |
|
pET-15b |
33ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
1ul |
|
2NEB Buffer |
4ul |
September 4th
Applied pET-15b and ATF1 to the blue gel electrophoresis.
No pET-15b was detected.
Extracted from Blue gel and purified ATF1.
September 11th
PCR reaction was carried out using the primer.
|
Buffer |
50ul |
|
dNTP |
20ul |
|
Primer mix |
1ul |
|
Genome DNA |
0.5ul |
|
KOD-FX |
2ul |
|
H2O |
26.5ul |
Electrophoresed in 1% agarose gel. Verified as ATF1.
Purified PCR products of ATF1.
Digested ATF1 and pET-15b with Xho1 and Bpu11021 at 37℃ for 2 hours.
|
ATF1 |
15.5ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
0.5ul |
|
2NEB Buffer |
2ul |
Added 1ul of BAP to the solution of pET-15b and incubated it at 37℃ for 30 minutes.
Ligated of pET-15b with ATF1 and transformed.
Applied pET-15b and ATF1 to the blue gel electrophoresis.
Isolated and purified them.
September 12th
Picked 32 colonies of ATF1 transformants.
September 13th
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in the LB in 3ml ampicillin(+) medium at 37℃ for 5 hours.
Miniprepped plasmid DNA.
Digested plasmid DNA with Xho1 and Bpu11021 at 37℃ for 2 hours.
|
ATF1 into pET-15b |
15.5ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
0.5ul |
|
2NEB Buffer |
2ul |
Applied pET-15b to the agarose gel electrophoresis.
No band was detected.
Plasmid DNA was digested with Xho1 and Bpu11021 at 37℃ (overnight).
|
ATF1 into pET-15b |
15.5ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
0.5ul |
|
2NEB Buffer |
2ul |
September 14th
ATF1 and pET-15b were Applied to electrophoresis to the agarose gel.
No band was detected.
Digested ATF1 and pET-15b with Bpu11021 at 37℃ overnight.
|
ATF1 |
26ul |
|
Bpu11021 |
1ul |
|
Bpu Buffer |
3ul |
|
pET-15b |
26ul |
|
Bpu11021 |
1ul |
|
Bpu Buffer |
3ul |
September 15th
Purified ATF1 and pET-15b.
Digested ATF1 and pET-15b with Xho1.
|
ATF1 |
26ul |
|
Xho1 |
1ul |
|
Bpu Buffer |
3ul |
|
pET-15b |
26ul |
|
Xho11021 |
1ul |
|
Bpu Buffer |
3ul |
pET-15b and ATF1 were applied to electrophoresis to the Blue gel and purified.
Ligated ATF1 with pET-15b and transformed them into E. coli cells.
September 16th
Picked 32 colonies of ATF1 transformants.
September 18th
Checked the colonies by colony cracking.
Picked up the colonies and cultured in 3ml LB medium with ampicillin at 37℃ (overnight).
September 19th
plasmid DNA was purified and digested with Xho1 and Bpu11021 at 37℃ for 2 hours.
|
ATF1 into pET-15b |
15.5ul |
|
Xho1 |
1ul |
|
Bpu11021 |
1ul |
|
BSA |
0.5ul |
|
2NEB Buffer |
2ul |
ATF1 and pET-15b were applied to electrophoresis to the agarose gel.
No band was detected.
Digested ATF1 and pET-15b with Xho1 and Blp1.
|
ATF1 |
15.5ul |
|
Xho1 |
1ul |
|
Blp1 |
1ul |
|
BSA |
0.5ul |
|
Buffer |
2ul |
|
pET-15b |
15.5ul |
|
Xho1 |
1ul |
|
Blp1 |
1ul |
|
BSA |
0.5ul |
|
Buffer |
2ul |
Added 1ul of BAP to the solution of pET-15b and incubated it at 37℃ for 30 minutes.
Applied pET-15b and ATF1 to the blue gel electrophoresis.
Isolated and purified them.
The ATF1 was ligated with pET-15b, and transformed into E. coli cells.
September 20th
Picked 64 colonies of ATF1 transformants.
September 21st
Checked the colonies by colony cracking.
Picked up colonies and cultured in the LB in 3ml LB medium with ampicillin at 37℃ (overnight).
September 22nd
Miniprepped plasmid DNA and digested it with Xho1 and Blp1 at 37℃ for 2 hours.
Applied ATF1 into pET-15b to the agarose gel electrophoresis.
No band was detected.
