Difference between revisions of "TSS Competent Cells"
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Wideloache (talk | contribs) m (Competent Cells - Small Scale moved to TSS Competent Cells) |
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| − | #Grow cells in ~4 mL LB until cloudy ( | + | #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 | ||
#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) | ||
| Line 11: | Line 11: | ||
#Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp. | #Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp. | ||
#Plate | #Plate | ||
| + | |||
| + | |||
| + | ==5X KCM== | ||
| + | #0.5M KCl | ||
| + | #0.15M CaCl<sub>2</sub> | ||
| + | #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
