Transformation problems

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Mon Aug 22 10:44:07 EST 1994


Dear Netters
I am having serious problems with some electroporations I am attempting.
The major problem is a rogue plasmid that is turning up everywhere, even 
in my negative control (E. coli cells alone - no plasmid). After 
electroporation these cells will grow on ampicillin plates, before 
electroporation they won't. The same plasmid is turning up in 'positive'
transformants from my ligations. I have checked everything I can think of
for contamination, the only thing left - and the most obvious source -
is the electroporation cuvettes although I find it hard to believe that
this plasmid has got into all of them! I have been reusing the cuvettes
after soaking 20 min. in 10% bleach then rinsing several times with
distilled water then 95% ethanol - can anyone tell me if this is 
sufficient to get rid of contaminating DNA? I have also sequenced part
of the rogue plasmid and it is a plasmid we are using in the lab (pET21)
but it has a large insert of foreign DNA that I don't recognize at all.
If anyone has any comments or suggestions on how to get rid of this
problem I would appreciate hearing from you. Incidentally, I did get
some of the correct transformants from two of my ligations but there
were also plenty of transformants with the wrong plasmid present. The
other two ligations have not yielded any correct transformants and I
am getting despreate!
Thanks in advance for your time
Emma
emma at hera.med.utoronto.ca



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