pcDNA3 with Wizard

[Paul N Hengen]

In article <1994Oct3.164057.1 at icrf.icnet.uk> g_watson at icrf.icnet.uk writes:

> I thought some fellow strugglers would like to hear my exprience using
> Promega's Wizard maxi-prep system.  I think its brilliant.  The yield is great
> and the UV trace looks wonderfull.  BUT I've tried it with Invitrogens pcDNA3
> and guess what,  it does not cut :-(.
> Well I faffed about for a while then hit on this really novel idea...
> Phenol; Phenol Cholorform IAA; Chorlorform IAA; Ether; Ethanol precipitation. 

Well then, so much for the kit ;-) Why bother with it? Just isolate plasmid DNA
by alkaline lysis and do all the organic extractions!

> All this after Promega's stuff.  Well the yield was lousy (about 30% of
> starting stuff :-( ).  But it did cut  :-).
> The odd part of this saga is that bluescript, when treated exatly
> the same as pcDNA (without the novel phenol and chloroform etc), cut perfectly
> with those same recalcitrant enzymes that would not cut the pcDNA without
> subsequent messing about.
> I hope in the future to discover which of the above steps is the one
> that removes whatever it is that is stopping my enzymes working.  The
> experimental design is quite complex and the grant application has already gone
> in  :-)  The real question however is what the hell is causing this most
> unco-operative behaviour.  I guess it must be a protein.

I'm very suspicious that the plasmids were extracted from different host
strains.  If this is still happening after transforming the plasmids into
the same host and then isolating DNA, I would be more willing to believe
there is something odd about the plasmid.....like mutations or deletions
in the restriction site region...or....or...maybe it's the....(gulp)...kit.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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