From Danijela Dukovski at Harvard Medical School. This protocol works well. --Julie Norville
DI water
10% Glycerol
Centrifuge
Ice water bath
Liquid nitrogen
Grow 500ml culture to OD 0.5 (approximately).
Spin down cells 5 times in ice cold 10% sterile glycerol.
Keep everything on ice and use a refrigerated centrifuge.
Each time you resuspend use a progressively smaller volume, and make sure all the cells are well resuspended. At the end resuspend in an appropriate volume. It should be pretty cloudy but not super dense. I just do it by eye, so it comes out about like the competent cells you buy.
Aliquot into Eppendorf tubes (I use 250-500uL, depending on how much I have and how lazy I am), freeze in liquid nitrogen, and store at -80.
How I usually do the washes: spin, resuspend in 250ml spin, resuspend in 100ml spin, resuspend in 50ml (can switch to Falcon tubes here) spin, resuspend in 25ml spin, resuspend in 10ml spin, final resuspension
Sometimes I cut one of these out depending on how much of a hurry I am in. I think the final volume (depends on the intial OD of your cells) usually ends up being 5-10mL, so you can get quite a few aliquots.
For transformation usually use 40-50uL of competent cells per transformation with a few ul (2-3) of ligation.