Difference between revisions of "TSS Competent Cells"
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Macampbell (talk | contribs) |
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#Centrifuge full speed for 30 sec, toss out supernatant | #Centrifuge full speed for 30 sec, toss out supernatant | ||
#Resuspend in 90uL of [http://openwetware.org/wiki/TSS TSS buffer] | #Resuspend in 90uL of [http://openwetware.org/wiki/TSS TSS buffer] | ||
− | #Add 10uL | + | #Add 10uL KCM |
#Spread about 5uL of untransformed competent cells to confirm that they are free of contamination. | #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) | #Add 1uL of DNA (use more [5uL] after a ligation) |
Revision as of 15:09, 17 October 2011
- 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.
- Plate
5X KCM
- 0.5M KCl
- 0.15M CaCl2
- 0.25M MgCl2