Difference between revisions of "TSS Competent Cells"

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Line 9: Line 9:
 
#Let sit on ice for 10min
 
#Let sit on ice for 10min
 
#Heat shock 90 sec at 42
 
#Heat shock 90 sec at 42
#Rescue for 1 hour in LB if antibiotic is not Amp.
+
#Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp.
 
#Plate
 
#Plate

Revision as of 23:16, 4 November 2008

  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 solution
  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