Saponin Lysis of RBCs

 

Electrocompetent Cells

From Danijela Dukovski at Harvard Medical School. This protocol works well. --Julie Norville

Materials

DI water
10% Glycerol
 

Special Equipment

Centrifuge
Ice water bath
Liquid nitrogen
 

Method

Grow 500ml culture to OD 0.5 （approximay）.

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.