low plasmid yields in dh5alpha

Jose de las Heras via methods%40net.bio.net (by josenet from tiscali.co.uk)
Wed Feb 20 19:09:12 EST 2008


"Ed Siefker" <ebs15242 from creighton.edu> wrote in message 
news:mailman.723.1203535541.2451.methods from net.bio.net...
> Hi.  We've been going through our old plasmids and making
> fresh stocks.  We've found a few that we're not able to
> miniprep and get reasonable concentrations (<50ng/ul).
> We're using Qiagen miniprep kits and invitrogen dh5alpha
> and have had much success purifying
>
> One thought was that the plasmids might be producing a
> product that is toxic to dh5alpha, and maybe they'd do
> better in another strain. Has anyone had a similar problem
> that was resolved by growing the plasmid in another strain?
>
> I'm thinking XL1-blue might help because it retains the
> lac repressor, and that might help stop any background
> expression from the lac promoter.  Any other ideas would
> be appreciated.  Thanks
> -Ed


this may be basic, but it can sometimes be overlooked...
especially in winter, the labs may get cold during the night etc... the 
lysis solution often starts to precipitate SDS, and your lysis won't be very 
effective unless you make sure the SDS gets back in solution (warm it up).

We recently had an episode where miniprep yields were low suddenly, and SDS 
precipitation was teh culprit. Not so easy to see sometimes in those white 
Qiagen bottles.

However, if you made several preps at one time and only some suffered, your 
problem is likely something else entirely.

Jose
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