Team:Paris Saclay/Notebook/September/8

Thursday 8th September

Lab work

Visualization

Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8

By Maxence

As no colonies were obtained after one night culture, a new Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th september) with the following protocol.

Concentration of insert and plasmid were determinated again by Nano drop. For each 20μl of reaction, mix the following reagents :

  • 0.65 µL of insert
  • 1.1 µL of plasmid
  • 8,25 µL of water
  • 10 µL of buffer mix

Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). Two different buffer mix were tested (the usual one and another one from Philippe). The PCR was performed as follow : 1 hour at 50°C.

Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson

By Maxence

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or containing controls (no buffer mix and plasmid alone) using the usual protocol. As mentioned before, two buffer mix were used. Furthermore, two different DH5a strains were tested (the usual one and another one from Philippe) and at least 12 transformants were cultured overnight : 3 (1 Gibson and 2 controls) x 2 buffer mix x 2 DH5a strains.

PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy

By Maxence

An other clonage strategy was performed by using Gibson between FKBP, gblock 2.2 (part of dCas9 NM - GFP 10) and pSB1C3 (the one from dCas9 ST - GFP 11) in order to have FKBP - GFP 10 in pSB1C3. For that purpose, 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)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of Q5 buffer (5X)
  • 0,5 µL of Q5 high fidelity polymerase
  • 32,5 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 $\infty$ 
Primers used were:
Matrix gblock FKBP in pJET clones 3, 4 and 5 gblock 2.2 in pUC19
Primers iPS145 and iPS146 iPS147 and iPS84
Tm 62°C 70°C
t 15 sec 15 sec

Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3

Maxence

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 7 min
Hold 4°C $\infinity\$ 
Primers used were:
Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers iPS83 and iPS84
Tm 63.6°C
t 1 min 30

After amplification, 3 µL of each 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)
dCas9 NM - GFP 10 - pSB1C3 3688
Result of the migration

GEL GEL GEL

Samples preparation for sequencing

by Maxence

20 µL of plasmides dCas9 ST - GFP 11 (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.