Lidstrom:Protocols: Difference between revisions

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==E. Coli==
==E. Coli==
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
==Electrocompetent Cells==
*You can make your own electro-competent cells for electroporation.
**From Amada's past mentor in undergrad:
***Grow the cells overnight
***Inoculate from this the next morning: generally use 200 uL/50 mL
***Let grow for about 3-4 h (OD 0.4-0.6 or so),  then centrifuge and wash 3 times with 10% glycerol (everything on ice).
***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
****Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care.  See [[Electrocompetent_Cells| this page]].  Would allow you to calculate efficiency.

Revision as of 17:56, 2 July 2012