Bulk prep problems

Beverly Trimpe TRIMPE at KIDS.WUSTL.EDU
Mon Nov 7 23:11:30 EST 1994


In <511877221wnr at genesys.demon.co.uk> Duncan at genesys.demon.co.uk writes:

> In article: <CyBC73.6uu at news.otago.ac.nz>  biocmag at sanger.otago.ac.nz (Michael Gieseg) writes:
> 
> > So it appears that even though the bugs are ampicillin resistant they conatain
> > no plasmid.  Is it possible that the plasmid has intergrated?

> How do you know that at the end of your growth the bugs are still Amp 
> resistant. My guess is that they have dumped the plasmid and you are just
> growing up E.coli! This is a major problem when using ampicillin. To 
> find out for sure plate an appropiate dilution of your culture 
> on LB and LB + Amp plates. The ratio will reveal all. If they are 
> still Amp and no plasmid then intergration is possibly the answer.
> If they have lost the plasmid ie Amp sensitive then you may have to 
> use Kan or CAT vectors. 
> Duncan
 ---------------------------------------------------------------------------- 
I recommend checking out the possibility of using carbenicillin.  We seem to
have crummy amp in our lab(plates go bad quickly). The carb lasts forever on
plates and IMHO gives your chosen bugs a better headstart. N.B. Its expensive!
Bev




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