Team:Paris Saclay/Notebook/September/6

Tuesday 6th September

Lab work

Visualization

Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19

By Maxence

Q5 PCR was performed on plasmids with the following protocol:

For each 50μl of reaction, mix the following reagents :

  • 1 µL of plasmid
  • 1 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 10 µL of Q5 buffer (5X)
  • 0,5 µL of Q5 high fidelity polymerase
  • 35,5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step Temperature Time
Initial denaturation 98°C 30sec
30 cycles 98°C 10sec
Tm 20sec
72°C t
Final Extension 72°C 2min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8 FRB in pJET clones 4 and 9 GFP 1.9 in pUC19
Primers iPS152 and iPS151 iPS149 and iPS150 iPS84 and iPS140
Tm 57,5°C 56,7°C 72°C
t 1 min 30 20 sec 30 sec

PCR Clean-up of PCR products

By Maxence

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

NanoDrop Measurements

By Maxence

Sample Concentration (ng/µL)
PCR fragment GFP 11 clone 6
 187.23
PCR fragment GFP 11 clone 8
 156.85
PCR fragment FRB clone 4
 75.67
PCR fragment FRB clone 9
 246.41
PCR fragment GFP 1.9
 22.06

Gel of cleaned up PCR products

By Maxence

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
GFP 11- pSB1C3 2500
FRB 374
GFP 1.9 862
Result of the migration

GEL GEL GEL

Linearization of PCR product GFP 11 - pSB1C3 from clone 8

By Maxence

PCR product GFP 11 - pSB1C3 has been linearized for further Gibson application by using DpnI treatment :

  • 30 µL of cleaned up PCR product GFP 11 - pSB1C3
  • 4 µL of fast digest buffer
  • 1 µL of DpnI
  • 5 µL of water

The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Transformation of DH5a cells with dCas9 NM - GFP 10 in PSB1C3 obtained by Gibson

By Maxence

Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (obtained by Gibson of fragment 1 PCR products (one with Q5 Pol and one with Phusion) + fragment 2 PCR products (one with Q5 Pol and one with Phusion) + pSB1C3), or containing Gibson control (no mix added when Gibson performed) using the usual protocol.