lysis after electroporation...
rhubner at gins.uia.ac.be
Wed Dec 16 16:54:44 EST 1998
if highest transformation efficiency is not the most important
objective, you can simply plate out the maximum volume of
electroporated cells immediately to continue your research...
I appreciate that you prepared electrocompetent cells to achieve
high rates, but some people find the method easier/robuster and
just electroporate all the time...
Perhaps this circumvents the problem, although I would be interested
to know what troubleshooting will pick up this new mystery...
Could it be that you try to transform one insert and that it is breaking
the beasts cell walls? I once PCRed up a 8kb fragment during the
Bacillus genome project and this could not be cloned in E. coli (even
in short bits!)... Cycle sequencing later revealed that this harbored an
enzyme that interferred with the construction of E. coli's cell wall...
All the best,
ps. BTW, because Mg++ and Tetracycline have antagonistic effects,
their concommittant use should be avoided, but I know of lot of
people that do not know this or anyway continue to use them together
for preparation of F' strains of E. coli... Paul (Hengen) has also a word
of caution in one of his TIBS columns about the combination TET and
electroporation, if I recall this properly...
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