Transforming chemically competent cells (Inoue)
- Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
- Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
- Incubate on ice for 30 minutes
- Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- Incubate cells for 30 seconds at 42°C.
- Incubate cells on ice for 2 min.
- Add 4 volumes of room temperature SOC (not critical)
- Incubate for 1 hour at 37°C on shaker.
- Note: Can also save some time here by reducing incubation to ~45 min.
- Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37°C.
First attempt varied several parameters: incubation time on ice prior to heat shock, heat shock length, addition of DTT at 20mM.
- DTT appeared to have little effect when added during transformation.
- Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
- Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
- Heat shock at 30 s or 60 s gave approximately similar results.
Achieved efficiency was 3 x 107 per microgram. A control transformation with Invitrogen cells was at 1.2 x 108 per microgram.