Thursday 25^th August

Lab work

Visualization

Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9

By Terrence

The extraction was carried out following the usual protocol.

The plasmid PSB1C3 with the insert : - SgRNA Nm were extracted from the clone 5 - Fragment 3 + 4 were extracted from the clone 6 - GFP1-9 were extracted from the clone 4

Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.

By Terrence

Extracted plasmids were digest with XbaI and PstI, following usual protocol.

Result of the digestion

Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation

By Mathilde

Q5 PCR was performed directly on gBlocks to amplify them following the protocol: Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents :

• 1µL of ligation product
• 1µ of dNTPs (10mM)
• 1µL of each primer mix (10µM)
• 10µL of q5 buffer
• 0,25µL of Q5 high fidelity polymerase
• 35,75µL of nuclease free water

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

Primers used were:

After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.

PCR products expected were :

Migration of gBlocks and ligation