Wednesday 20^th July

Lab work

Biobrick characterization

pclTAA, pclTAG and pclTq transformations

By Laetitia and Caroline

In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pclTAA, pclTAG and pclTq. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Streptomycin (50µg/ml) and incubated overnight at 37°C.

Pre-culture of BL21

By Laetitia and Caroline

We picked an unique colony from a petri dish and soaked it in medium of LB (4mL). Then we incubated it at 37°C with agitation at 180 rpm overnight.

Extraction of plasmid DNA

By Naiane and Terrence

We use the protocol described in the experiment tab in order to extract the K1372001 plasmid.

We took 1.5 mL of overnight culture to use at the beggining of the protocol. At the end, the DNA was resuspended with 50 µL of TE/RNase.

Visualization

High fidelity PCR on bacteria transformed with pPS16_005

By Alice

High fidelity PCR on bacteria transformed with pPS16_005 did not get PCR products. We supposed that there was a problem with iPS83 primer dilution. That is why we performed again this PCR after diluting again theses primers. We followed the same protocol as previously. iPS83 and iPS126 primers were used. Annealing temperature was 72°C, and initial step was prolonged to 7 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.

PCR products expected were :

We did not observed PCR products. This is maybe due to a iPS83 primer default. New primers iPS83 will be ordered to perform again this PCR.

Low Fidelity Dreamtaq PCR of newly transformed DH5α with pPS16_007

By Mathilde

The transformed pPS16_007 in DH5α (from 19.07.2016) showed blue and white colonies and were then amplified on PCR with the DreamTaq polymerase following the usual protocol with Tm at 57°c and 5min for the initial denaturation.

We divided up the PCR mix in 6 PCR tubes and added in each one a different clone from transformed pPS16_007 culture. The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).

However the transformed pPS16_002 and pPS16_004 did not present any blue colony, so they were not amplified on PCR. Those heat shock transformations were conducted again this day.

Results :

PCR products expected were :

The electropheresis on agarose gel showed no PCR product for pPS16_007 clones 1 and 2, only pPS16_007 clones 3 and 5 seem to present a good size band.

A PCR of transformed pPS16_007 was made again with the exact same protocol but with 5 different clones picked from the culture.

We put those PCR products on electropheresis on agarose gel in addition to the previous pPS16_007 clones 3 and 5. Only clones 3, 5 and 11 were at the good size.

Culture of bacteria transformed with pPS16_002 and pPS16_004

By Alice

After transformation made on the 19/07/16, bacteria were spread on petri dishes without working XGal and IPTG, explaining that we get white colonies only. These colonies were spread again on petri dishes with LB, Ampicilin (50µg/mL), XGal (0.25µL/mL) and IPTG (0.1µL/mL).

Get DNA Closer

Electrophoresis of PCR products DS_SPcasN, DS_TDcasN and pZA21

By Caroline

PCR products obtained the 19/07/2016 were put to migrate for 30min in a 0,8% agarose gel.

pZA21 transformation

By Laetitia and Caroline

In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pZA21. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Kanamycin (50µg/ml) and incubated overnight at 37°C.

Pre-culture of DS_SPcasN and DS_TDcasN

By Laetitia and Caroline

For each Cas (SP and TD), we picked an unique colony from a petri dish and soaked it in medium of LB (4mL) and spectinomycin (2.6 µL).

Then we incubated it at 37°C with agitation at 180 rpm overnight.