TSS Competent Cells

From GcatWiki
Jump to: navigation, search
  1. Grow cells in ~4 mL LB until cloudy (OD600 = 0.5)
  2. Put on ice
  3. Transfer 1mL into an eppendorf tube on ice, let cool
  4. Centrifuge full speed for 30 sec, toss out supernatant
  5. Resuspend in 90uL of TSS buffer
  6. Add 10uL KCM
  7. Spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
  8. Add 1uL of DNA (use more [5uL] after a ligation)
  9. Let sit on ice for 10min
  10. Heat shock 90 sec at 42
  11. Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp.
  12. Plate


  1. 0.5M KCl
  2. 0.15M CaCl2
  3. 0.25M MgCl2