Preparing chemically competent cells
- Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
- Centrifuge for 10 minutes at 3000 rpm and 4oC.
- Remove supernatant and replace with 10% original volume TSS.
- Make 100 uL aliquots and store at − 80oC.
- Thaw TSS cells on ice.
- Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
- Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
- Let sit for 30 minutes on ice.
- Incubate cells for 30 seconds at 42oC.
- Add 1 mL SOC (2XYT is also suitable) at room temp.
- Incubate for 1 hour at 37oC.
- Plate 200 µL onto plate with appropriate antibiotic.
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL