TSS Competent Cells
Revision as of 12:51, 19 October 2011 by Macampbell
- Grow cells in ~4 mL LB until cloudy (OD600 = 0.5)
- Put on ice
- Transfer 1mL into an eppendorf tube on ice, let cool
- Centrifuge full speed for 30 sec, toss out supernatant
- Resuspend in 90uL of TSS buffer
- Add 10uL KCM
- Spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
- Add 1uL of DNA (use more [5uL] after a ligation)
- Let sit on ice for 10min
- Heat shock 90 sec at 42
- Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp.
- 0.5M KCl
- 0.15M CaCl2
- 0.25M MgCl2