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From 2014.igem.org

Contents 1 Friday 18th July 1.1 Lab work 1.1.1 1 - Transformation of supercompetent cells with CaCl2 1.1.2 2 - Plasmid DNA Purification 1.1.3 2 - Preparation of electrocompetent DY330 and transformation via pJBEI6409

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

Protocol

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

1. Dilute 1 mL of DY330 in 100mL of LB at 30°C.
2. When optical density is around .6, divide the 100mL into four 50mL centrifuge tubes. Centrifuge for four minutes at 4°C, 4000rpm. Discard supernatant.
3. Repeat, but this time with 25mL of 10% glycerol solution in each tube.
4. Repeat, but this time with 12.5mL.
5. 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

1. 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.
2. Electroporation at 2500V, 132W, 40µF.
3. Add 950 µL of cold LB in each cuvette.
4. Incubate at 30°C and under agitation for two hours.
5. 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.

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