Team:KIT-Kyoto/Notebook/YJL

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YJL

13th August

We designed the PCR primers to amplify YJL068C gene.



19th August

  1. Make Primer mix

We added 102.2 uL and 100.3 uL H2O to the forward and reverse primers, respectively, to make 100 pmol/microL solutions.

We obtained 10 uL of each primer solution and added 80 uL H2O to make the 10 pmol/microL primer mix.


  1. PCR

We performed PCR to amplify the YJL068C gene.

Buffer

50ul

dNTP

20ul

Primer mix

1ul

Genome DNA

0.5ul

KOD-FX

2ul

H2O

26.5ul

(Annealing

52°C)

We detected two DNA bands around 1400bp and 3000bp.

(The correct band is 1400bp.)

We changed annealing temperature to 54°C or 57°C and tried PCR again.

We detected a correct size DNA band around 1400bp.

The clear DNA band was amplified at 54˚C of annealing temperature.

We performed PCR to amplify YJL068C gene at 54°C of annealing temp (total volume was 500 microL).












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 Ban2 to check whether PCR succeeded or not.

We ligated YJL068C and pSB1C3.

YJL068C

24ul

M Buffer

3ul

BSA

1ul

Xba1

1ul

Spe1

1ul


YJL068C

6ul

H Buff.

2ul

H2O

11ul

BanⅡ

1ul


We digested psb1c3 with XbaI and SpeI at 37°C for 90 minutes.

pSB1C3

49ul

M Buffer

5.78ul

BSA

1ul

Xba1

1ul

Spe1

1ul

We added 1uL 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 microL) 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 1uL BAP to it.

pSB1C3

96ul

Buffer

11ul

BSA

1ul

Xba1

1ul

Spe1

1ul

We electrophoresed psb1c3 it in 1% Blue gel.

It was extracted from the Blue gel.

YJL068C

5ul

pSB1C3

5ul

Ligation Mix

10ul

We added 5 microL 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 Pst1 and EcoR1, and incubated at 37°C for 90 minutes.

Plasmid DNA

10ul

H Buffer

2ul

H2O

6ul

EcoR1

1ul

Pst1

1ul

We applied them to 1% agarose gel electrophoresis and checked them.

5 samples showed the correct size.


2.

We digested pSB1C3 with BsiW1.

pSB1C3

10ul

3 NEB Buffer

2ul

BsiW1

1ul

H2O

7ul



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 containing Chloramphenicol.

Plasmid DNA was purified by miniprep.



24th August

We added 29.5uL of H2O to purified YJL068C.

We digested YJL068C with BanII and checked.

YJL068C

5ul

H Buff.

2ul

H2O

12ul

BanⅡ

1ul

We detected the correct size of DNA band.

We digested YJL068C with SpeI and BamHI.

YJL068C

24.5ul

Buffer

3ul

BSA

0.5ul

Spe1

1ul

BamH1

1ul

We digested pET-15b with XbaI and BamHI and dephosphorylated with 1uL BAP.

pET-15b

87ul

Buffer

10ul

BSA

1ul

Xba1

1ul

BamH1

1ul

We extracted DNA from the Blue gel.

We added 10 uL 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 containing ampicillin.

We added 20ul of IPTG to them and incubated.

Cell free extract was prepared and applied to SDS-PAGE to detecte ATF2 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

24ul

Buffer

3ul

BSA

1ul

Xho1

1ul

Nde1

1ul


pET-15b

33ul

Buffer

4ul

BSA

1ul

Xho1

1ul

Nde1

1ul


DNA samples were applied to 1% Blue gel electrophoresis and then purified.

We added 10 uL 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 ATF1 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 XhoⅠand NdeⅠ overnight.

YJL068C

24ul

Buffer

3ul

BSA

1ul

Xho1

1ul

Nde1

1ul


pET-15b

33ul

Buffer

4ul

BSA

1ul

Xho1

1ul

Nde1

1ul




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

10ul

pET-15b

10ul

Ligation Mix

10ul

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 XhoⅠand NdeⅠ(overnight).

YJL068C

24.5ul

Buffer

3ul

BSA

0.5ul

Xho1

1ul

Nde1

1ul


pET-15b

24.5ul

Buffer

3ul

BSA

0.5ul

Xho1

1ul

Nde1

1ul

Applied YJL068C and pET-15b(prepared on September 5th) to the blue gel electrophoresis.

Isolated and purified it.

Ligation of pET-15b with YJL (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

15.5ul

Buffer

2ul

BSA

0.5ul

Xho1

1ul

Nde1

1ul

(2)

BsiW1

1ul

Buffer

2ul

Plasmid DNA

17ul

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 XhoⅠand BlpⅠ .

YJL068C

24.5ul

NEB 4 Buffer

3ul

BSA

0.5ul

Xho1

1ul

Blp1

1ul


pET-15b

24.5ul

NEB 4 Buffer

3ul

BSA

0.5ul

Xho1

1ul

Blp1

1ul

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 ampicillin(+) medium.



September 22nd

Miniprepped plasmid DNA(YJL068C into pET-15b) and digested it.

Plasmid DNA

16ul

Xho1

1ul

Blp1

1ul

NEB 4 Buffer

2ul

Electrophoresed in 1% agarose gel.

We could not get any YJL068C into pET-15b E. coli cells.