(none)

Fraser Dr N J njf16154 at ggr.co.uk
Wed Aug 7 10:49:41 EST 1996


Sailesh.Surapureddi at mcb.liu.se (Sailesh Surapureddi) wrote:
>
>
>Hi  Everybody
>
>        Here is the ice cold transformation protocol.. I am posting this
>for the benifit all transformers.
>
>
>1. Pre warm the plates @37 (very imp) for 30min or 60min which ever is
>convient to you.
>
>2. Chill the tranformation tubes (falcon 2059 or clean eppies, the later
>takes more time to chill)
>
>3. Thaw your competent cell on ice waterbath.
>
>4. Open them on ice only and aliquot to the pre chilled tubes (if they are
>from Life tech, I usually take 25ul per tube).
>
>5. Add the DNA, (if its ligated DNA, make sure to clean it with ethanol
>pption and compeltely dry it).
>
>6. I routinely take 2-4uL of DNA (or ligated mix i.e 1/4th of the 20ul
>ligation mix) for 25 -50uL of competent cells.
>
>7. Add slowly with gentle swirl, with the pipette tip.
>
>8. Allow it to stay on ice for 3-5min.
>
>9. Take the entire amount and spread  on the prewarmed LB +antibiotic
>plates  (no SOC etc etc).
>
>10. Incubate for 10-12hrs @37C.
>


But how did you make the cells competent in the first place ?!




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