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Team:KIT-Kyoto/Notebook/YJL
From 2013.igem.org
YJL068C
13th August
We designed the PCR primers to amplify YJL068C gene.
19th August
1. Make Primer mix
We added 102.2 µL and 100.3 µL H2O to the forward and reverse primers, respectively, to make 100 pmol/µL solutions.
We obtained 10 µL of each primer solution and added 80 µL H2O
to make the 10 pmol/µL primer mix.
2. PCR
We performed PCR to amplify the YJL068C gene.
|
Buffer |
50µL |
|
dNTP |
20µL |
|
Primer mix |
1µL |
|
Genome DNA |
0.5µL |
|
KOD-FX |
2µL |
|
H2O |
26.5µL |
(Annealing : 52°C)
We detected two DNA bands around 1400bp and 3000bp.
(The correct band is 1400bp.)
We changed annealing temperature to 54°C and 57°C and tried PCR again.
We detected a correct size DNA band around 1400bp.
The clear DNA band was amplified at 54˚C (annealing temperature).
We performed PCR to amplify YJL068C gene at 54°C of annealing temp (total volume was 500 µL).
20th August
1
We performed PCR to check whether the primers can work or not.
(Annealing temperature: 55°C)
We detected a DNA band of the correct size around 900bp.
2
We purified the YJL068C gene (PCR was performed on 8/19).
We digested YJL068C with BanII to check whether PCR succeeded or not.
We ligated YJL068C and pSB1C3.
|
YJL068C |
24µL |
|
M Buffer |
3µL |
|
BSA |
1µL |
|
XbaI |
1µL |
|
SpeI |
1µL |
|
YJL068C |
6µL |
|
H Buff. |
2µL |
|
H2O |
11µL |
|
BanII |
1µL |
We digested pSB1C3 with XbaI and SpeI at 37°C for 90 minutes.
|
pSB1C3 |
49µL |
|
M Buffer |
5.78µL |
|
BSA |
1µL |
|
XbaI |
1µL |
|
SpeI |
1µL |
We added 1µL BAP to dephosphorylate the pcb1c3 gene.
We electrophoresed the YJL068C gene and pSB1C3 (digested for ligation) in 1% Blue gel.
We saw the clear band of YJL068C, and not so clear band of pSB1C3.
They were extracted from the Blue gel.
We prepared the spare the spare in case of failure (made stocks of purified YJL068C and pSB1C3).
We purified YJL068C and pSB1C3 (these were in one tube) and ligated them.
We transformed it all (20 µL) into E.coli cells.
21th August
1
We checked the YJL068C gene (digested with Ban2 8/20) by electrophoresing.
We could see correct band (succeeded PCR).
2
We checked of the insert by colony cracking (transformed 8/20).
We couldn’t see clear bands, but cultured furthermore.
3.
We made the spare in case of failure (prepared 8/20).
We purified the YJL068C gene (digested 8/20).
We digested pSB1C3 with XbaI and SpeI (90 minutes at 37°C), and added 1µL BAP to it.
|
pSB1C3 |
96µL |
|
Buffer |
11µL |
|
BSA |
1µL |
|
XbaI |
1µL |
|
SpeI |
1µL |
We electrophoresed pSB1C3 it in 1% Blue gel.
It was extracted from the Blue gel.
|
YJL068C |
5µL |
|
pSB1C3 |
5µL |
|
Ligation Mix |
10µL |
We added 5 µL H2O to dried YJL068C gene and pSB1C3 each.
We ligated them and transformed it into E.coli cells.
22th August
1.
We did miniprep to purify DNA.
We digested YJL068C in pSB1C3 with PstI and EcoRI, and incubated at 37°C for 90 minutes.
|
Plasmid DNA |
10µL |
|
H Buffer |
2µL |
|
H2O |
6µL |
|
EcoRI |
1µL |
|
PstI |
1µL |
We applied them to 1% agarose gel electrophoresis and checked them.
5 samples showed the correct size.
2.
We digested pSB1C3 with BsiWI.
|
pSB1C3 |
10µL |
|
NEB Buffer 3 |
2µL |
|
BsiWI |
1µL |
|
H2O |
7µL |
23th August
1.
We applied YJL068C in pSB1C3 (prepared on 8/22) to 1% agarose gel electrophoresis.
We detected the correct size of DNA.
Each transformant was cultured in LB liquid medium with chloramphenicol.
Plasmid DNA was purified by miniprep.
24th August
We added 29.5µL of H2O to purified YJL068C.
We digested YJL068C with BanII and checked.
|
YJL068C |
5µL |
|
H Buff. |
2µL |
|
H2O |
12µL |
|
BanII |
1µL |
We detected the correct size of DNA band.
We digested YJL068C with SpeI and BamHI.
|
YJL068C |
24.5µL |
|
Buffer |
3µL |
|
BSA |
0.5µL |
|
SpeI |
1µL |
|
BamHI |
1µL |
We digested pET-15b with XbaI and BamHI and dephosphorylated with 1µL BAP.
|
pET-15b |
87µL |
|
Buffer |
10µL |
|
BSA |
1µL |
|
XbaI |
1µL |
|
BamHI |
1µL |
We extracted DNA from the Blue gel.
We added 10 µL H2O to dried YJL068C gene and pET-15b.
We ligated them and transformed it into E.coli cells.
25th August
We cultured them furthermore.
26th August
We checked the insertion of DNA by colony cracking.
We cultured each transformant in LB liquid medium with ampicillin.
We added 20ul of IPTG to them and incubated.
Cell free extract was prepared and applied to SDS-PAGE to detect YJL068C protein.
29th August
Cell free extract was prepared with FastBreak Cell Lysis Reagent and applied to SDS-PAGE.
30th August
We performed PCR to amplify YJL068C.
(Annealing temperature: 55°C)
31th August
We purified the YJL068C (performed PCR at 8/30).
We digested YJL068C with BanII to check whether PCR succeeded or not.
We ligated YJL068C and pSB1C3.
We digested YJL068C with XhoI and NdeI for ligation.
We purified pET-15b and digested with XhoI and NdeI.
|
YJL068C |
24µL |
|
Buffer |
3µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
|
pET-15b |
33µL |
|
Buffer |
4µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
DNA samples were applied to 1% Blue gel electrophoresis and then purified.
We added 10 µL H2O to dried YJL068C gene and pET-15b.
We ligated them and transformed it into E.coli cells.
September 1st
Picked 32 colonies of YJL068C transformants.
September 4th
Digested YJL068C and pET-15b overnight.
Applied pET-15b and YJL068C to the blue gel electrophoresis.
Isolated and purified it.
September 5th
Purified PCR products and dissolved 24ul of H2O.
Purified pET-15b.
Digested YJL068C and pET-15b with XhoI and NdeI overnight.
|
YJL068C |
24µL |
|
Buffer |
3µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
|
pET-15b |
33µL |
|
Buffer |
4µL |
|
BSA |
1µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
September 6th
1.
Applied YJL068C and pET-15b(prepared on September 5th) to the blue gel electrophoresis.
Isolated and purified it.
Ligation of pET-15b with YJL 068C (prep).
|
YJL068C |
10µL |
|
pET-15b |
10µL |
|
Ligation Mix |
10µL |
Transformed YJL068C into pET-15b E. coli cells.
2.
Purified PCR products and pET-15b were dissolved in 24.5ul of H2O.
YJL068C and pET-15b were digested with XhoI and NdeI(overnight).
|
YJL068C |
24.5µL |
|
Buffer |
3µL |
|
BSA |
0.5µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
|
pET-15b |
24.5µL |
|
Buffer |
3µL |
|
BSA |
0.5µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
Applied YJL068C and pET-15b(prepared on September 5th) to the blue gel electrophoresis.
Isolated and purified it.
Ligation pET-15b and YJL068C (prep).
Transformed YJL068C into pET-15b E. coli cells.
September 7th
Picked 48 colonies of YJL068C transformants.
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in 3ml LB medium with ampicillin.
September 8th
Plasmid DNA was prepared by miniprep and digested with XhoI/NdeI.
(1)
|
Plasmid DNA |
15.5µL |
|
Buffer |
2µL |
|
BSA |
0.5µL |
|
XhoI |
1µL |
|
NdeI |
1µL |
(2)
|
BsiWI |
1µL |
|
Buffer |
2µL |
|
Plasmid DNA |
17µL |
Electrophoresed in 1% agarose gel.
Not succeeded.
September 15th
Transformed YJL068C into pET-15b E. coli cells.
September 16th
Picked 48 colonies of YJL068C transformants.
September 17th
Checked the colonies by colony cracking.
Picked up the colonies and cultured in 3ml LB medium with ampicillin.
September 18th
Miniprepped plasmid DNA(YJL068C into pET-15b) and digested it.
Electrophoresed in 1% agarose gel.
Not succeeded.
September 19th
YJL068C and pET-15b were digested with XhoI and BlpI.
|
YJL068C |
24.5µL |
|
NEB Buffer 4 |
3µL |
|
BSA |
0.5µL |
|
XhoI |
1µL |
|
BlpI |
1µL |
|
pET-15b |
24.5µL |
|
NEB Buffer 4 |
3µL |
|
BSA |
0.5µL |
|
XhoI |
1µL |
|
BlpI |
1µL |
Transformed YJL068C into pET-15b E. coli cells.
September 20th
Picked 64 colonies of YJL068C transformants.
September 21st
Checked the colonies by colony cracking.
Picked up the appropriate colonies and cultured in the LB in 3ml medium with ampicillin.
September 22nd
Miniprepped plasmid DNA(YJL068C into pET-15b) and digested it.
|
Plasmid DNA |
16µL |
|
XhoI |
1µL |
|
BlpI |
1µL |
|
NEB Buffer 4 |
2µL |
Electrophoresed in 1% agarose gel.
We could not get any YJL068C into pET-15b E. coli cells.
