p.j. van santbrink
santbrin at CHEM.LEIDENUNIV.NL
Mon Aug 19 08:06:56 EST 1996
Hi, fellow netters
I recently started a new project and I have a few questions. I
transformed the E. coli strain MC1061/P3 with the eucaryotic expression
vector pCMD8. The transformation went well, and in the next step I want
to isolate large amounts of plasmid. I've had success with the plasmid
mini/midi kit from QIAGEN so I want to use that kit for the isolation.
But because pCDM8 is a low-copy-number plasmid containing the COLE1
origin of replication I have to use large volumes to obtain fair amounts
of plasmid. The problem is that we have but one SORVALL GSA rotor and
that means long waiting hours to get a ride. The enclosed QIAGEN handbook
comes with a solution: use chloramphenicol. But they don't give much
extra information. So my question is:
- does somebody have experience with chloramphenicol stimulated plasmid
isolation with the QIAGEN-kit and if so could he/she send me a protocol?
A second question:
- the strain MC1061/P3 already containts the P3 episome (which to my
knowledge is a 57 kb plasmid); will this plasmid also be purified with
pCDM8 using the QIAGEN kit?
I hope there's a person on this planet who can help me.
p.s.: my E-mail address is: santbrin at lacdr.LeidenUniv.nl
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