jmollerup at mcb.molbio.ku.dk
Fri Jul 14 04:41:53 EST 2000
When we do electrotransformation of E.coli (SURE or JM109 cells) we get a
low tranformation efficiency (3 x 10E5) which certainly is lower than could
The cells are harvested at OD550 0.8, washed twice in milliQ water +10%
glycerol (kept at 4C all the time) and frozen in liquid nitrogen, stored
Invitrogen 0.2cm cuvettes, BioRad Gene Pulser II at 2.5 kV, 200 ohm, 25
microFahrad, 80 microL cells + 1 microL DNA - low salt (e.g., pBD-GAL4), E.
coli and cuvettes are kept on ice! Then growth at 37C in NY for 1hr before
plating to ampicillin plates.
We have tried several times with different E.coli (and preparations thereof)
but the transformation efficiency is low and useless. Does anyone have an
opinion of what to do or what we do wrong?
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