Difference between revisions of "TSS Competent Cells"
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| − | #Grow cells in ~4 mL LB until cloudy (OD<sub>600</sub>=0.5) | + | #Grow cells in ~4 mL LB until cloudy (OD<sub>600</sub> = 0.5) |
#Put on ice | #Put on ice | ||
#Transfer 1mL into an eppendorf tube on ice, let cool | #Transfer 1mL into an eppendorf tube on ice, let cool | ||
| Line 15: | Line 15: | ||
==5X KCM== | ==5X KCM== | ||
#0.5M KCl | #0.5M KCl | ||
| − | #0.15M | + | #0.15M CaCl<sub>2</sub> |
| − | #0.25M | + | #0.25M MgCl<sub>2</sub> |
Latest revision as of 14:51, 19 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
