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)


 * 1) inoculate 10 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm
 * 2) inoculate 1 L LB-light in 2-litre-flask with 10 mL preculture
 * 3) incubate until OD600 0,4-0,6 (~5 h)


 * from now on everything is done at 2-4°C (best in a cold room)


 * 1) cool 1L-culture 10-15 minutes in ice water (shake sometimes)
 * 2) divide culture into 4 cooled 250 mL bins for centrifugation
 * 3) centrifuge 20min @ 2°C, 4200 rpm
 * 4) discard supernatant
 * 5) resuspend pellet in 5 mL bidest H2O
 * 6) add bidest H2O up to 250 mL
 * 7) centrifuge 20min @ 2°C, 4200 rpm
 * 8) discard supernatant immediately
 * 9) resuspend pellet in residual supernatant
 * 10) add bidest H2O up to 250 mL
 * 11) centrifuge 20min @ 2°C, 4200 rpm
 * 12) discard supernatant immediately
 * 13) resuspend pellet in residual supernatant
 * 14) transfer suspension in 50 mL Falcons
 * 15) add 10% Glycerol up to 50 mL
 * 16) centrifuge 10 min @ 2°C, 4000 rpm
 * 17) discard supernatant
 * 18) estimate volume of the pellet; fill up with equal volume of 10% Glycerol
 * 19) resuspend pellet on ice; ‘’’don´t vortex!!’’’ (just shake cautiously)
 * 20) divide cells into 200 or 400 µL Allicots (use 1,5 mL Eppis)
 * 21) freeze in liquid N2 or dry-ice
 * 22) store @ -80°C

Transformation via electroporation

 * 1) add 0,5-2 µL plasmid to 50 µl electrocompetent cells
 * 2) electroporate at U=2,5 kV, C= 25 µF, R = 200 Ώ
 * 3) transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37°C
 * 4) centrifuge 2 min @ 800 rpm and plate on selective LB-Medium