Team:KIT-Kyoto/Notebook/YJL

13th August

We designed the PCR primers to amplify YJL068C gene.

 

 

19^th 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/uL solutions.

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

 

2.    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 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 uL).

 

 

 

 

 

 

 

 

 

 

 

20^th 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	24uL
M Buffer	3uL
BSA	1uL
XbaI	1uL
SpeI	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
XbaI	1uL
SpeI	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 uL) 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
XbaI	1uL
SpeI	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 uL 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	10uL
H Buffer	2uL
H2O	6uL
EcoRI	1uL
PstI	1uL

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

5 samples showed the correct size.

 

2.

We digested pSB1C3 with BsiWI.

pSB1C3	10uL
NEB Buffer 3	2uL
BsiWI	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 with 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
SpeI	1uL
BamHI	1uL

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

pET-15b	87uL
Buffer	10uL
BSA	1uL
XbaI	1uL
BamHI	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 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	24uL
Buffer	3uL
BSA	1uL
XhoI	1uL
NdeI	1uL

 

pET-15b	33uL
Buffer	4uL
BSA	1uL
XhoI	1uL
NdeI	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 1^st

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 5^th

Purified PCR products and dissolved 24ul of H2O.

Purified pET-15b.

 

Digested YJL068C and pET-15b with XhoI and NdeI overnight.

YJL068C	24uL
Buffer	3uL
BSA	1uL
XhoI	1uL
NdeI	1uL

 

pET-15b	33uL
Buffer	4uL
BSA	1uL
XhoI	1uL
NdeI	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 XhoI and NdeI(overnight).

YJL068C	24.5uL
Buffer	3uL
BSA	0.5uL
XhoI	1uL
NdeI	1uL

 

pET-15b	24.5uL
Buffer	3uL
BSA	0.5uL
XhoI	1uL
NdeI	1uL

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 7^th

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 8^th

Plasmid DNA was prepared by miniprep and digested with XhoI/NdeI.

(1)

Plasmid DNA	15.5uL
Buffer	2uL
BSA	0.5uL
XhoI	1uL
NdeI	1uL

(2)

BsiWI	1uL
Buffer	2uL
Plasmid DNA	17uL

Electrophoresed in 1% agarose gel.

Not succeeded.

 

 

September 15^th

Transformed YJL068C into pET-15b E. coli cells.

 

 

September 16^th

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 18^th

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

Electrophoresed in 1% agarose gel.

Not succeeded.

 

 

 

 

 

September 19^th

YJL068C and pET-15b were digested with XhoI and BlpI.

YJL068C	24.5uL
NEB Buffer 4	3uL
BSA	0.5uL
XhoI	1uL
BlpI	1uL

 

pET-15b	24.5uL
NEB Buffer 4	3uL
BSA	0.5uL
XhoI	1uL
BlpI	1uL

Transformed YJL068C into pET-15b E. coli cells.

 

 

September 20^th

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 22^nd

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

Plasmid DNA	16uL
XhoI	1uL
BlpI	1uL
NEB Buffer 4	2uL

Electrophoresed in 1% agarose gel.

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