Quint Lab:electrocompetent cells e.coli

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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