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.