Thursday 11^th August

Lab work

Visualization

Transformation of DH5a cells with pPS16_020

By Léa

Dh5a cells were transformed with pPS16_020 (GFP 1-9 ligated with pSB1C3 digested), or the linearized pSB1C3 (control), or not transformed (control) using the usual protocol.

pPS16_010 extraction

By Alice

After colony screening PCR, 3 clones (clones 3, 7 and 8) seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel.

Band size expected:

Migration of FRB plasmid

Nano Drop:

Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 11) and pPS16_009(clone 7)

By Terrence

The extraction was carried out following the usual protocol.

Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7)

Nano Drop:

High Fidelity Phusion PCR of transformed cell with ligation 2 and 3

By Laetitia

Phusion PCR was performed on ligation product 2 (gblock 2.1 ligated with gblock 2.2) and ligation product 3 (gblock 3.1 ligated with gblock 3.2) made on 09/08/16. It was done following this protocol. For the PCR on ligation 2 the primers used were IPS 123 and IPS 84. For the PCR on ligation 3 the primers used were IPS 128 and IPS 83.

Gel1

DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC 19

By Léa and Laetitia

PCR DreamTaq was performed on 6 clones of each transformed cells from 10/08/16. Each clone was plated and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel.

Phusion PCR on PSB1C3

By Alice

PSB1C3 was amplified with Phusion DNA polymerase following this protocol. Two primers (iPS41 and iPS42) were chosen. Annealing temperature was 70.9°C. Elongation step lasted 1min. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.

PCR products expected:

Migration of PSB1C3 amplification

Result of the ligation 2-3 PCR.

By Caroline

We made the PCR again as the previous result wasn't satisfying. Ligation product were cultivate overnight.

Result of the ligation 2-3 PCR

Bringing DNA closer

Extraction of DS-SPcasN- and DS-TDcasN-

By Terrence

The extraction was carried out with the Nucleobond Ax Kit.

Extraction of DS-SPcasN- and DS-TDcasN-

Nano Drop: