"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
"I don't got to show you no stinking .signature..."

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