Wednesday 20th 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.
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 :
| Plasmids
|
Band size (bp)
|
| pPS16_005
|
964
|
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.
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.
