Day 1
- Thaw competent cells on ice ~5min
- Add 50μl of competent cells to 1μl plasmid or 5μl of ligation mix (no more than 10% ligation mix final volume) in a PCR tube
- Incubate on ice 30min
- Heat-shock in PCR machine 42°C 45s (no heated lid)
- Place 2min on ice
- Add 100μl LB
- Recover 45-60min 37°C. When using antibiotics which do not target the ribosomes (e.g. Ampicillin etc.) this step can be skipped
- Plate on plates containing appropriate antibiotics. Take along a negative control as well.
- Grow O/N upside down 37°C
Day 2
- Pick several colonies, screen by colony PCR
- Grow correct clones in 5ml LB in 15ml falcon 37°C 220RPM O/N
Day 3
- Make glycerol stock and isolate plasmid
- Sequence