"Crud" in plasmid preparation

Michael Coady coady at ERE.UMontreal.CA
Thu Sep 2 00:45:50 EST 1993


Hello Netland,
	I hope somebody has some ideas on this one.  I've been purifying
a plasmid library that was grown in suspension in low-melt agarose, and
I've found that there is some contaminant that I can't get rid of.  I
spin the bacteria out of the agarose, and get rid of leftover agarose
when I do my alkaline lysis.  I get a fair amount of DNA, too, but there
is some kind of gummy substance that stays in the wells when I do agarose
electrophoresis and it traps most of the DNA.  I tried using glassmilk
(actually, Bio-Rad's Prep-A-Gene) and it had no effect.  I tried several
phenol-CHCl3 extractions but no effect.  I tried using Proteinase K for
several hours but it had no effect.  I thought it might be contaminating
DNA from the bacteria but cutting it with a restriction enzyme had no
effect.  I tried a method for precipitating down agarose by adding 1/10
volume of 4 M LiCl after phenol extraction but there was no effect.  I
have no idea what this gummy substance could be nor why it is so tenacious
and would appreciate any ideas you have.

Mike Coady

-- 
Michael J. Coady
COADY at ERE.UMONTREAL.CA
"I don't got to show you no stinking .signature..."



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