Team:GO Paris-Saclay/labnotebook-folc

folC part

Ligation of gBlock-folC in pGEMT easy

17/07 – Kenn

pGEMT easy

2ul

T4 Buffer 10X

1 ul

folC 1.8kb

10 ng/ul

1.5 ul

15 ng

T4 ligase

1 ul

Water

qsp 10 ul

4.5 ul

2 h at room temperature

Streaking clones: gBlock-folC  pGEMT easy

18/07 – Kenn

Collection of 5 white clones with insert were streaked on LB Xgal IPTG Amp.

 Extraction of the pGEMTeasy- FolC plasmid

20/07 –  Yueying (?)

Clone 1, 2 and 4 from 2 ml of culture were used for this extraction with the extraction kit.

Nanodrop results :

Clone 1

40,2ng/µl

Clone 2

19,67ng/µl

Clone 4

26,43ng/µl

 

digestion of the pGEMTeasy- FolC plasmid

20/07 –  Yueying (?)

Plamid was digested by NotI fast digest

Clone 1

Clone 2

Clone 4

Plasmid DNA µL

5

7

6

Fast digest green buffer

1

1

1

Water µL

3,5

1,5

2,5

NotI µL

0,5

0,5

0,5

30 min at 37°C

digestion pGEMTeasy-folC by NotI results:

expected length : 2 bands at 3000 and 1000 pb

  • Clone 1 : not the expected length
  • Clone 2 – 4 : not digested

PCR of pGEMTeasy- FolC plasmid

23/07 –  Yueying

In order to explain what was obtained with this digestion, we did a PCR with pGEMteasy plasmid as control.

Cycle step

Protocol

Cycles

Temp.

Time

Initial denaturation

95°C

2min

1

Denaturation

95°C

30s

30

Annealing

44°C

30s

Extention

72°C

2min30

1min/Kb

Final extension

72°C

5min

1

 Mix below divided in 4 tubes : 1,2 and 4 with DNA extract from clone 1,2 and 4 and tube 4 with plasmid control

Dream Taq polymerase

0,5 µL

 Taq green buffer

10 µL

Water

69 µL

M13 rev

10 µL

M13F-20

10 µL

dNTP

0,8 µL

PCR Results:

Expected size :

  • plasmid + insert = 2 Kb
  • plasmid = 230 pb

Our control shows a signal stripes at 2,5 Kb which is weird

2 and 4 signal stripes are as expected but quite wick.

Digestion of the PCR product

23/07 –  Yueying

We digest the PCR product 2 and 4 with BamHI

2

4

  DNA µL

10

10

Fast digest green buffer

2

2

Water µL

17

17

BamHI µL

1

1

30 min at 37°C

digestion PCR product 2 and 4 by BamHI results:

Digestion failed, we can’t conclude anything on clone 2 and 4

pGEMt easy- folC PCR fusion

24/07 –  Yueying

Cycle step

Protocol

Cycles

Temp.

Time

Initial denaturation

98°C

2min

1

Denaturation

98°C

30s

30

Annealing

55°C

30s

Extention

72°C

2min30

1min/Kb

Final extension

72°C

10min

1

fusion polymerase

0,5 µL

  5X buffer HF

10 µL

Water

29,5 µL

Primer A 10 µM

2,5 µL

Primer B 10 µM

2,5 µL

dNTP

4 µL

DNA

1 µL

PCR results :

Expected size :

  • plasmid + insert = 2Kb
  • plasmid = 230pb

We can see a band at 2Kb, we obtain our plasmid with folC

Gel extraction of pGEMt easy- folC

We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit

Nanodrop results:

pGEMt-easy-FolC

12 ng/µL

Digestion of pGEMt easy- folC

24/07 –  Yueying

There is nothing about this digestion on drive

PCR colony

24/07 –  Xavier

PCR COLONY ON 10 WHITE COLONIES TRANSFORMED WITH pGEMTeasy FOLC BY XAVIER 23-24/07?? -> missing

Extraction of PCRBlunt-folC

06/08 –  Clémence

Clone 2,5,7 and 8 were used for this extraction of PCRBlunt-FolC with miniprep protocol.

folC has been ligated in PCRBLUNT

Nanodrop results

Clone

Concentration (ng/µL)

2

13,5

5

13,9

7

10,4

8

21,4

Digestion of PCRBlunt-folC

06/08 –  Clémence

Plasmid was first digested by XbaI and PSTI in order to separated FolC from the plasmid and then digested by SmaI in order to degrade pCR Blunt.

Buffer 2.1

4 µL

XbaI

1 µL

PstI

1 µL

DNA

30 µL

Water

4 µL

XbAI and PSTI were inactivated during 20min at 80°C before we added 1µL of SmaI for the third digestion

https://lh4.googleusercontent.com/eFONIpNSQ4UwxIFKURQqVcp-ecQsrrXsPgA5OanAw-9ShFI_GWG6H9Kw4H1e-dn7Vsy_t84Ay8Vk3R-PidYtJ1AV5qGQpnE2SVDNlaEbS4bx1Q4VnkTXUZ6_vBdGoB0qHAyXlduy

Gel extraction of folC

06/08 –  Clémence

We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit

Nanodrops results:

Clone

Concentration (ng/µL)

2

21

5

17,6

7

13

8

13

Digestion of PSB1C3 plasmid

06/08 –  Clémence

In order to ligate folC in PSB1C3, we first digested this plasmid with XbaI and PstI.                                                                                                                                                          

Buffer 2.1

4 µL

XbaI

1 µL

PstI

1 µL

DNA

15 µL

Water

9 µL

Size of digest plasmid = 2044pb

Gel extraction of digested PSB1C3

06/08 –  Clémence

We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit

Nanodrops results:

Digested PSB1C3

48,4 ng/µL

ligation of folC in PSB1C3

07/08 –  Clémence

For clone 2 and 5 :

Digested PSB1C3

1,5 µL

folC

6,5µL

T4 DNA Ligase

1 µL

Buffer T4 Ligase

1 µL

For clone 7 and 8 :

Digested PSB1C3

1 µL

folC

7µL

T4 DNA Ligase

1 µL

Buffer T4 Ligase

1 µL

Transformation of DH5alpha with ligation mix

08/08 –  Britany

We used the heat shock transformation protocol.

100 µL of each transformation were then spread into petri dish.

Few clone were obtain.

PCR colony

08/08 –  Britany

Cycle step

Protocol

Cycles

Temp.

Time

Initial denaturation

94°C

5min

1

Denaturation

95°C

30s

30

Annealing

49°C

30s

Extention

72°C

2min30

1min/Kb

Final extension

72°C

10min

1

DNA

22,4 µL

Taq

0,1 µL

Buffer green Taq

3 µL

dNTP

2 µL

Oligo 1 VR

1,25 µL

Oligo 2 VF2

1,25 µL