chloramphenicol

[John Dixon]

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.

snip

>         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,
>                         Bernard


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
Qiagen preps. 

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
crud.

> [no affiliations]

likewise
John


> 
> 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
Cambridge University    
United Kingdom                           e-m: jpcd0 at mole.bio.cam.ac.uk