Preparing Competent Cells for transformation

[rasing02]

I want to prepare chemically competent cells for BL21DE3 ( PLySs) cell line, can someone please email me or upload the protocol for the same, my Id is rasing02@gmail.com, I also wanted to know the protocol for preparing competent cells varies for different cell lines of Ecoli? or remains the same, meaning will it be different for Dh5alpha versus BL21DE3 ( PLySs) , I would really appreciate a quick response.

[CharonY]

I assume you mean calcium competent cells (there are different types). The basic protocol is the same for basically all strains, though there are modifications. Most of the time they appear to be more rooted in lab culture rather than real-life increase in efficiency.

I do not know the precise protocol off the top of my head, but I am pretty sure you can find it quickly via a simple search. For more details I would advise reading Sambrook's Molecular cloning.

 

The basic premise is always growing the cells to the log-late log phase, harvest, keep cool. Then wash with CaCl2 solution containing glycerol, pellet cells again, and resuspend in reduced volume, and quickly freeze them.

[Greippi]

My protocol is similar to the above. I've used it successfully for S17 and DH5alpha, as mentioned it'd be pretty much the same for other strains.

 

Grow cells until they're in log phase (0.6 absorbance at 600nm)

*keep the cells on ice/as cool as possible throughout the procedure*

Spin cells down, obtain pellet

Resuspend in 0.1M MgCl2 (I use 20ml)

Spin down, obtain pellet

Resuspend in 0.1M CaCl2 (I use 20ml)

Spin down, obtain pellet

Resuspend in 20% glycerol, 0.1M CaCl2 (I use 1ml)

Aliquot.

Snap freeze with liquid nitrogen.

Store at -80C

 

I've gotten better at making competent cells, it takes practice, - the first batch of S17s I made worked, but took a little longer than usual to grow up after transformation.

Edited November 26, 2010 by Greippi