- prepare a fresh overnight culture in LB
- inoculate 100 ml of prewarmed SOC media with 1 ml of the overnight
culture, shake until an OD600 = 0.5 is reached (approx. 2 h)
- cool the culture on ice, collect cells by spinning at 5000 rpm
for 5 to 10 min (4°C)
- discard the supernatant, supend the pellet in cold TFB1 buffer
(first gently with a glass pipette in a few ml only, then dilute to 30 ml)
- inculate on ice for 90 min
- collect the cells (5 min, 5000rpm, 4000xg, 4°C
- dicant the supernatant, gently resuspend cells in 4 ml of TFB2
solution
- prepare aliquots (100-200 µl) and freeze in liquid nitrogen
- store the cells at -70°C
all steps should be performed in the cold room with cold solutions, pipettes,
tubes
Transformation
- thaw cells on ice
- gently mix cells with cold DNA (upto 10µl in 100µl
cells)
- incubate on ice for 20 min
- heat-shock cells for 90s at 42°C
- add 500-900µl of SOC medium and incubate for 60 min at
37°C (shake for highest transformation efficiency)
- plate appropriate volume on selective LB plates, incubate plates
overnight at 37°C
solutions:
TFB1: 100mM RbCl
50 mM MnCl2
30 mM K-acetate
10 mM CaCl2
15% Glycerol (w/v)
pH 5,8
TFB2: 10 mM MOPS
10 mM RbCl
75 mM CaCl2
15 % Glycerol (W/v)
pH 6,8 (with KOH)
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