Tuesday 6^th September

Visualization

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

By Maxence & Mahnaz

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:

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 :

Result of the migration

No PCR products were obtained for GFP 1.9. Concerning FRB and GFP 11 - pSB1C3 amplification, good results were obtained and clones 9 for FRB and clone 8 for GFP 11 - pSB1C3 were selectionned.

Linearization of PCR product GFP 11 - pSB1C3 from clone 8

By Maxence & Mahnaz

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 (pPS16_016) obtained by Gibson

By Maxence & Mahnaz

Dh5a cells were transformed with pSB1C3 containing dCas9 NM - GFP 10 (pPS16_016) (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 control (no mix added when Gibson performed) using the usual protocol.