jpcd0 at mole.bio.cam.ac.uk
Tue Aug 20 06:24:02 EST 1996
In article <1996Aug19.202224.5834 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) wrote:
> 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
> >vector pCMD8.
> 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.
Hi Bernard, I have had some hassles with these strains for supF selection:
whose recommendations do you follow - the ones in Invitrogens catalog? I
still have problems with revertants, mainly because the plasmids I have
contain a wild type amp gene, so I only have tet selection. I have now
switched to cat plasmids to get over this. What concentration of
antibiotics do you plate onto, especially if you are using cocktails of
more than one?
> 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).
DH10B:p3 from Gibco is endA-, and more rec deficient.
> >- 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,
I use this strain, and DH10B:p3, with Terrific broth and always see a
little of the p3 episome in my digests, especially if they are amber
revertants that contain only p3. This is after simple alkaline lysis or
I think the p3 comes through because unlike the bacterial DNA it is not
bound to the cell wall and therefore does not precipitate with all the
> [no affiliations]
> Bernard Murray, Ph.D.
> bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
John Dixon Lab 44 (1223) 334131
Wellcome/CRC Institute Fax 44 (1223) 334134
Department of Genetics
United Kingdom e-m: jpcd0 at mole.bio.cam.ac.uk
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