Team:Paris Saclay/Notebook/July/18
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from Bacterial Culture made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/17 17th July] | from Bacterial Culture made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/17 17th July] | ||
- | ===2 - | + | ===2 - Preparation of electrocompetent DY330 and transformation via pJBEI6409=== |
''by Sean'' | ''by Sean'' | ||
- | + | '''''A. Preparation of DY330 strain''''' | |
- | + | ||
- | ''''' | + | |
- | + | ||
Protocol | 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. | ||
Revision as of 08:41, 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.