Quick Competent Cells

Items needed:
ependorphs
cold 10% glycerol
cold H2O
overnight culture on plates or in medium



Quickie competent cells

Resuspend a loopful of cells off a plate in 1mL cold sterile water
Wash 2 times with cold water
Wash 1 time with cold 10% glycerol
Resuspend in 50microL cold glycerol

Wash = centrifuge ~1min at 13,000x, decant supernatant




No-so-quickie Competent cells

Innoculate 3mL LB with 100microL overnight culture
Shake vigorously for ~4 hours
Spin down cells (~1min at 13,000x) - to do in ependorph, do 1.5 mL, add the rest and spin again
Decant supernatant

Repeat below 3 times:
Keep cold!
Add 1mL cold 10% glycerol 
Resuspend cells
Spin down (~1min, 13,000x) - use a centrifuge in the cold room, so everything stays cold!
Decant supernatant

Add 90microL 10% cold glycerol for final volume
Aliquot 40microL to 2 tubes for 2 electroporations