Thursday 8^th September

Lab work

Visualization

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

By Maxence & Mahnaz

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 (pPS16_019) obtained by Gibson

By Maxence & Mahnaz

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), 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.

Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy

By Maxence & Mahnaz

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 (pPS16_018). 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:

Primers used were:

PCR on GFP 1.9 in pUC19 with 3% DMSO

By Maxence & Mahnaz

As the annealing temperature (Tm) seems too high to obtain good results for GFP 1.9 in pUC19 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, 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 buffer (5X)
• 0,5 µL of Phusion polymerase
• 31 µL of nuclease free water
• 1.5 µL of DMSO

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

Primers used were:

PCR Clean-up of PCR products

By Maxence & Mahnaz

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

NanoDrop Measurements

By Maxence & Mahnaz

Gel of cleaned up PCR products

By Maxence & Mahnaz

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 :

Migration of FKBP plasmid

GEL 4 : FKBP








All PCR products were at the good size.

Migration of FKBP plasmid

GEL 5 : gblock 2.2








All PCR product were at the good size.

Migration of FKBP plasmid

GEL 6 : gfp 1.9