Template:SBB-Protocols SmallScaleCompetent: Difference between revisions
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(New page: #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 90u...) |
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#Grow cells in ~4 mL LB until cloudy (OD600=0.5) | #Grow 30uL of cells in ~4 mL LB until cloudy (OD600=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 | ||
Latest revision as of 11:43, 1 July 2010
- Grow 30uL of 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 solution
- Add 10uL KCM
Step 6.5: do the negative control before adding the DNA
- Add 1uL plasmid DNA
- Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute, rescue 1 hr, then incubate and/or plate
- Always do negative controls on your competent cell prep!
Sector a region of your plate and spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
