pcDNA3 with Wizard

g_watson at icrf.icnet.uk g_watson at icrf.icnet.uk
Mon Oct 3 11:40:57 EST 1994

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. 
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.
	 Anyone have any ideas or similar problems?
		Graham Watson

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