Difference between revisions of "TSS Competent Cells"
From GcatWiki
Wideloache (talk | contribs) |
Wideloache (talk | contribs) |
||
| Line 3: | Line 3: | ||
#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 TSS | + | #Resuspend in 90uL of [http://openwetware.org/wiki/TSS TSS buffer] |
#Add 10uL [http://gcat.davidson.edu/GcatWiki/index.php/KCM_Preparation KCM] | #Add 10uL [http://gcat.davidson.edu/GcatWiki/index.php/KCM_Preparation 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. | ||
Revision as of 07:19, 17 December 2008
- 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
