Tuesday 12th July
Lab work
Bringing DNA closer
Amplification of DS-TDcasN-, DS-SPcasN- and pZA21 plasmids
By Caroline
Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the get DNA closer tool and the pZA21 plasmid in which the tool will be inserted. A PCR was carry out using Q5® High-Fidelity 2X Master Mix and following the usual protocol adapted to have 50µL at the end.
Electrophoresis of the PCR amplification
By Caroline
5µL of the amplifications are mixed with 1µL of purple loading dye. The solutions are put into a 0.8% agarose gel and migrated for 30min.
Migration of SP, TD and pZA21
Liquid culture of transformed NM, TD, ST1 and SP cas9
By Mathilde and Laetitia
Two colonies from each petri dishes were cultured in 1mL of LB and Spectinomycin (50µg/mL).
Our 8 cultures were put in incubation at 37°c, 180 rpm ON.
Biobrick characterization
Electrocompetents and transformation by electroporation of BL21
By Charlene
Cells transformed on 11/07/16 grew overnight. However no colony was observed.
To control if there is a bacteria transformed which was not put on Petri dish, we performed another experiment. 100 µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions :
- 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL)
- 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL)
- 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL)
Cells were incubated for 8h at 37°C, 200 RPM.
No bacteria grew so we concluded that there was a problem with the transformation.
Visualization
Transformation of gBlock 4.2 pPS16_008
By Laetitia and Mathilde
DH5α heat-shock competent cells were transformed with plasmid pPS16_008 (gBlock 4.2).
Petri dishes peparation (x3) :
- 10µL of Ampicillin (50µg/mL)
- 5µL of Xgal (0.25µL/mL)
- 2µL of IPTG (0.1µL/mL)
- 20mL of LB+Agar
Cells transformed with the two gBlocks constructions were plated in duplicata (50µL and 150µL) and a control construct was plated alone.
The plates were put in incubation at 37°c overnight.
High fidelity PCR on bacteria transformed with pPS16_004 and pPS16_007
By Alice
A PCR with Q5® High-Fidelity 2X Master Mix was performed on clones selected after screening PCR of the 11/07/16, following this protocol.1151_pheoR and 1152_pheoF primers were used. Annealing temperature was 60°C and initial denaturation was prolonged to 5 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 30min at 100V.
PCR products expected were :
| Plasmids
|
Band size (bp)
|
| pPS16_004
|
865
|
| pPS16_007
|
763
|
No PCR products were observed. The reason remains a mystery.
gBlock sequencing
By Caroline
PCR products obtained 11/07/16 : pPS16_001, pPS16_002, pPS16_003, pPS16_005,pPS16_006, pPS16_007, pPS16_008 and pPS16_009 were sent to sequencing.
