Electro competent Escherichia coli cells
this protocol works fine with E.coli DB3.1 cells
Materials:
- 10 mL LB-Medium (1% Bacto Trypton; 0,5 % Yeast Extract; 0,5% NaCl)
- 1L LB-light Medium (1% Bacto Trypton; 0,5 % Yeast Extract; 0,25% NaCl)
- 2L cooled bidest H2O
- 200 mL cooled, sterile-filtered 10% Glycerol
- box with ice-water for 2-litre-flask
- 4 pre-cooled 250 mL (or 2x500 mL) bins for centrifugation
- 2 pre-cooled 50 mL Falcons
- centrifuge pre-cooled to 2°C (max. 4°C)
- inoculate 10 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm
- inoculate 1 L LB-light in 2-litre-flask with 10 mL preculture
- incubate until OD600 0,4-0,6 (~5 h)
- from now on everything is done at 2-4°C (best in a cold room)
- cool 1L-culture 10-15 minutes in ice water (shake sometimes)
- divide culture into 4 cooled 250 mL bins for centrifugation
- centrifuge 20min @ 2°C, 4200 rpm
- discard supernatant
- resuspend pellet in 5 mL bidest H2O
- add bidest H2O up to 250 mL
- centrifuge 20min @ 2°C, 4200 rpm
- discard supernatant immediately
- resuspend pellet in residual supernatant
- add bidest H2O up to 250 mL
- centrifuge 20min @ 2°C, 4200 rpm
- discard supernatant immediately
- resuspend pellet in residual supernatant
- transfer suspension in 50 mL Falcons
- add 10% Glycerol up to 50 mL
- centrifuge 10 min @ 2°C, 4000 rpm
- discard supernatant
- estimate volume of the pellet; fill up with equal volume of 10% Glycerol
- resuspend pellet on ice; ‘’’don´t vortex!!’’’ (just shake cautiously)
- divide cells into 200 or 400 µL Allicots (use 1,5 mL Eppis)
- freeze in liquid N2 or dry-ice
- store @ -80°C
Transformation via electroporation
- add 0,5-2 µL plasmid to 50 µl electrocompetent cells
- electroporate at U=2,5 kV, C= 25 µF, R = 200 Ώ
- transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37°C
- centrifuge 2 min @ 800 rpm and plate on selective LB-Medium
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