Team:Paris Saclay/Notebook/July/18
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=Friday 18th July= | =Friday 18th July= | ||
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==Lab work== | ==Lab work== | ||
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===1 - Transformation of CaCl<sub>2</sub> supercompetent cells=== | ===1 - Transformation of CaCl<sub>2</sub> supercompetent cells=== | ||
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''by Arnaud & Romain'' | ''by Arnaud & Romain'' | ||
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===2 - Plasmid DNA Purification=== | ===2 - Plasmid DNA Purification=== | ||
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''by Fabio'' | ''by Fabio'' | ||
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===2 - Preparation of electrocompetent DY330 and transformation via pJBEI6409=== | ===2 - Preparation of electrocompetent DY330 and transformation via pJBEI6409=== | ||
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''by Sean'' | ''by Sean'' | ||
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# Incubate at 30°C and under agitation for two hours. | # 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. | # 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. | ||
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'''Members present''': | '''Members present''': | ||
* Instructors and advisors: Solenne. | * Instructors and advisors: Solenne. | ||
* Students: Arnaud, Fabio, Mathias, Romain and Sean. | * Students: Arnaud, Fabio, Mathias, Romain and Sean. | ||
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Revision as of 08:49, 7 August 2014
Contents |
Friday 18th July
Lab work
1 - Transformation of CaCl2 supercompetent cells
by Arnaud & Romain
Strains transformed: MG1655, MG1655Z1. from Bacterial Cultures made the 17th July
2 - Plasmid DNA Purification
by Fabio
- BBa_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.
Members present:
- Instructors and advisors: Solenne.
- Students: Arnaud, Fabio, Mathias, Romain and Sean.