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Team:Aix-Marseille/Protocol:supercompetent ecoli cells
From 2014.igem.org
Supercompetent Escherichia coli cells
- Streak E.coli cells on an LB plate with or without antibiotics.
- Allow cells to grow at 37°C overnight.
- Place three or four colonies in 5 mL LB media (+ antibiotic selection if necessary), grow overnight at 37°C.
- Dilute the preculture to OD600 = 0.005 in 200 mL LB.
- Allow cells to grow at 37°C (250 rpm), until OD600= 0.5 (about 2-3 hours).
- Transfer cells to 4 centrifuge bottles (50 mL), and place cells on ice for 20 minutes.
- Centrifuge cells in Sorval GSA rotor at 4°C for 10 minutes at 3 500 rpm. Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room.
- Discard the supernatant and resuspend cells in 80 mL of cold Tfb1 (300 mM KOAc, 0.05 M MnCl2, 0.1 M CaCl2, Glycerol 15%).
- Centrifuge cells using Sorval RT6000B rotor at 4°C for 5 minutes at 3 500 rpm.
- Discard the supernatant and resuspend cells (by pipetting) in 8 mL of cold Tfb2 (0.01 M NaMOPS pH7, 0.075 M CaCl2, 0.01 M KCl, Glycerol 15%). Incubate on ice for 15 minutes. Transfer 500 µL of cells into (1.5 mL) Ependorff tubes placed on ice. Cells stored at -80°C can be used for transformation for up to ~6 months.
- Add 1,5 ng of plasmide DNA to 100 µl of competent cells at 4°C.
- Incubate the mixture on ice for 30 minutes.
- Transfer the reaction to a 42°C water bath for exactly 1 minute and 30 seconds.
- Add 900 µL of LB medium to each tube and incubate at 37°C for 1 hour to allow cells to recover and express the antibiotic resistance marker.
- Centrifuge cells for 1 minute at 6 000 rpm. Discard 900 µL of the supernatant and resuspend cells. Spread cells on selective media.
- Incubate all plates overnight at 37°C.
Preparation of supercompetent cells
Transformation
