Wednesday 24^th August

Visualization

Gel Electrophoresis of Gibson Assembly's products(fragment 3-4) and GFP 1-9 cloned in PSB1C3

By Terrence

Colony PCR of 12 colonies transformed with Gibson Assembly products (fragment 3-4).

Colony PCR of 12 colonies containing the GFP 1-9.

pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction

By Terrence

The extraction was carried out following the usual protocol.

Result of the extraction

Nano drop :

Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET

By Terrence

First we made a ligation with

Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol.

Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.

Plasmid DNA extraction (Midi Prep)

By Léa & Manhaz

We have decided to use the plasmid coding dCas9 Nm to amplify the fragement corresponding g-block 1.2. 200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.

The plasmid then reserved in -20.

PCR Q5 on plasmid

By Léa & Manhaz

Plasmid pJet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2. also we amplify the plasmids extracted from 2 colonies contrain the plasmid coding dCas Nm ordered from adgene.

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:

Result of migration

NanoDrop Measurements

By Mahnaz

gBlock 1.1 and gblock 1.2 Ligation

By Mahnaz

gBlock 1.1 and gblock 1.2 were ligated together as following :

• 8µL of gBlock 1.1 PCR product from the 04/08/2016
• 8µM of gblock 1.2 PCR product from 24/08/2016
• 2µL of Buffer T4 10X
• 2µL of ligase T4 enzyme

The ligation product was put at 4°c overnight.