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 :

Result of the migration

The three different samples of segment 1 (ligated pPS16_001 and pPS16_002) and segment 2 (ligated pPS16_001 and pPS16_002) PCR product seem to be at the expected size. But segment 2 is in insufficient quantity, so the segment 2 ligation and purification will be conducted again.

pPS16_003 and pPS16_004 Ligation

By Mathilde

gBlocks pPS16_003 and pPS16_004 were ligated together as following :

• 4µL of pPS16_00 PCR product from the 08/08/2016
• 4µM of pPS16_004 PCR product from 08/08/2016
• 1µL of Buffer T4 10X
• 1µL of ligase T4 enzyme

The ligation product was put at 4°c overnight.

PCR Colony

By Mahnaz

We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying