Dwindling Plasmid yield *HELP*

David Huen s.d.huen at bham.ac.uk
Tue Apr 26 06:32:46 EST 1994


In article <leach-200494114012 at med-pharm5.bu.edu>, leach at mbcrr.harvard.edu
(Martin Leach) wrote:
> 
> Dear netters,
> 
> Ok, here's the situation. I've got a couple of important deletion
> constructs (containing a piece of a promoter) in luciferase reporter
> constructs (promega).
> 
> In the promoter-luciferase constructs (no SV40 enhancers) i am finding
> several constructs have very little yield after maxiprepping. I use the
> Qiagen tip method - (i know it is not the columns or solutions as other
> plasmid preps come out fine while some consistently come out crap!
> 
> I have tried re-transforming the bad plasmids into bacteria, but so far no
> joy. Someone else in the lab is having a problem with this
> promoter-luciferase construct also (different promoter though).
> 
> 
> If anyone can suggest anything that may help I would greatly appreciate it.
> 
> aTdHvAaNnKcSe
> 
> Martin
> 
> 
> p.s. I am gonna try terrific broth vs. Luria Broth (we usually use this), i
> am also going to increase ampicillin from 25ug/ml to 100ug/ml.

I use very similar constructs myself and have been usually getting good
yields at 100 ug/ml Amp.

For me, the major reason for poor yields is, I think, outgrowth of either
contaminating non-transformed bugs (which are present in your colony after
transformation). So I routinely restreak before inoculating into a
bulkprep. When, inoculating, I pick a single colony into 2-3 mls TB+Amp AND
IMMEDIATELY pour that into 200-400 mls of TB+Amp which is then incubated at
37 till I'm satisfied (ie. bother to come in to work next day). I do this
to maximise the exposure time of the bugs to Amp before the beta-lactamase
from the resistant population wipes it out from the medium. This has given
me much more reliable yields that in the past.

David Huen, Insitute of Cancer Studies, Univ. of Birmingham
P.S. Sure the prob is not related to Bolo? :) luv, Gronk.



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