Preparation of electrocompetent cells

Michael Kawalek kawalekm at earthlink.net
Fri Nov 19 10:49:54 EST 2004



>Hello everybody
>
>I find it difficult to obtain high transformation efficiencies (>10^9cfu/µg)
>using electrocompetent cells (E. coli - TG1) prepared by the standard
>protocols (Sambrook & Russel/Current protocol...).
>
>What is the pitfall of the standard protocol? How can I easily optimise the
>efficiency?
>
>Alternatively:  Does anybody know an alternative, "foolproof", method that
>really gives highly competent cells?
>
>
Hello
I know a few "tricks" that seems to improve results a log or so.  It seems that the cells are very
very sensitive to contamination in centrifuge bottles (probubly traces of soap residue).  Use new
bottles that haven't used for any other application.  Autoclave your bottles completely full of
nano-pure water and dump after autoclaving.  After pelleting the cells in the centrifuge, rotate the
bottles 90 degrees and spin for a few seconds to pull the residue solute contaminted water away from
the pellet.  When resuspending the pellet in your nano-pure water, keep the bottle constantly almost
submerged in an ice-slurry bath.  Be very patient.  Don't lift the bottle out of the ice to check
it's status.  

Give this a try and see how much your transformation effecieny improves.
Michael




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