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Team:Paris Saclay/Notebook/July/18
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==Lab work== | ==Lab work== | ||
| - | ===1 - Transformation of supercompetent cells with | + | ===1 - Transformation of supercompetent cells with CaCl<sub>2</sub>=== |
''by Arnaud & Romain'' | ''by Arnaud & Romain'' | ||
Revision as of 20:18, 21 July 2014
Contents |
Friday 18th July
Lab work
1 - Transformation of supercompetent cells with CaCl2
by Arnaud & Romain
Strains transformed: MG1655, MG1655Z1. from Bacterial Cultures made the 17th July
2 - Plasmid DNA Purification
by Fabio
- J23119 (x2)
from Bacterial Culture made the 17th July
2 - Preparation of electrocompetent DY330 and transformation via pJBEI6409
by Sean
A. Preparation of DY330 strain
Protocol
- Dilute 1 mL of DY330 in 100mL of LB at 30°C.
- When optical density is around .6, divide the 100mL into four 50mL centrifuge tubes. Centrifuge for four minutes at 4°C, 4000rpm. Discard supernatant.
- Repeat, but this time with 25mL of 10% glycerol solution in each tube.
- Repeat, but this time with 12.5mL.
- Remove supernatant and resuspend pellet with any remaining supernatant. Collect all of the strain from the four tubes then store in ice.
B. Transformation by electroporation
Protocol
- Fill three electroporation cuvettes as follows. a)50µL of DY330+2µL of pJBEI6409 plasmid. b)50µL of DY330+4µL of pJBEI6409. c)Control without DNA.
- Electroporation at 2500V, 132W, 40µF.
- Add 950 µL of cold LB in each cuvette.
- Incubate at 30°C and under agitation for two hours.
- Spread on nine petri dishes (LB+Cm): 50 µL from each cuvette, 100 µL from each cuvette, remainder from each cuvette. Incubate overnight at 30°C. NB: before spreading the remainder, centrifuge it, then remove 750 µL of supernatant.
People there:
- Instructors and advisors: Solenne.
- Students: Arnaud, Fabio, Mathias, Romain and Sean.
