bernard at elsie.nci.nih.gov
Mon Aug 19 15:22:24 EST 1996
In article <9608191312.AA13154 at chembul.leidenuniv.nl>,
santbrin at CHEM.LEIDENUNIV.NL says...
>I transformed the E. coli strain MC1061/P3 with the eucaryotic expression
>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
>The enclosed QIAGEN handbook comes with a solution: use chloramphenicol.
I've not found the need to use chloramphenicol amplification with
MC1061/P3, at least when isolating pcDNAI which is a slightly modified
version of pCDM8, and get quite reasonable yields, especially
from rich media - the copy number isn't *that* low. So, maybe try
moving to Terrific Broth or Super Broth and make sure the culture
is well shaken.
The only tip that I have is to stick to the recommended amounts
of ampicillin (or carbenicillin etc.) and tetracycline. You can add
kanamycin to the cocktail if you are worried that the host strain is
not maintaining the P3. Don't forget that the strain does not have
the endA mutation which means that all minipreps must be cleaned up
(eg. phenol/chloroform) before digesting them. For the same reason I
don't recommend the Promega Wizard kits for purification as they do
not separate the DNA from the nuclease (I have no experience with
QIAGEN kits with this strain).
Anothe possibility would be to move the insert to a more
modern high copy expression vector (my personal favourite is pCI-neo).
>- 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 believe that the size of the P3 episome means that it is
precipitated with the bacterial genomic DNA during alkaline lysis
conditions. Also, from what I understand, the episome is only
present as a single copy in each cell and so would only represent
a small fraction of non-chromosomal DNA within the cell. Again, I
have no practical experience with the QIAGEN kits with this strain.
I hope this is of some use,
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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