Preparing chemically competent cells
Revision as of 09:39, 16 January 2006 by Barry Canton (Chemically competent cells moved to Preparing chemically competent cells)
- Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
- Centrifuge for 10 minutes at 3000 rpm and [math]4^o[/math]C.
- Remove supernatant and replace with 10% original volume TSS.
- Note: Qualitative experience shows better results if aliquots are made in the [math]4^o[/math]C room.
- Note: It is best if you keep the cells on ice once they are resuspended in TSS and keep tubes that you aliquot them into on ice.Kathleen
- Make 100 μl aliquots and store at [math]-80^o[/math]C.
- 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.
- Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- Incubate cells for 30 seconds at [math]42^o[/math]C.
- Add 1 mL SOC (2XYT and LB are also suitable) at room temp.
- Incubate for 1 hour at [math]37^o[/math]C on shaker.
- Note: Can also save some time here by reducing incubation to ~45 min.
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37 °C.
- Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL
Filter sterilize (0.22 μm filter) and store at 4˚C or -20˚C.