SS Phagemid gradients, KI or CsCl?

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Jul 22 21:56:22 EST 1992


In article <1992Jul21.154049.25431 at usenet.ins.cwru.edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>
>We're trying to purify a mg or so of phagemid DNA, and need it to be free
>from contaminating double stranded DNA, such as might come from broken
>cells.  The protocol (sketchy) we're following suggests using EtBr/KI
>gradients for this, and collecting the lower band (SS) and discarding the
>upper band.  Our first go at this was a bust, as we lost almost all of the
>DNA in the work up. (the KI solution containing the DNA was sucked up into
>the H2O-saturated N Butanol!)
>
>I measured the density of the KI solution (1.6) and it is the same as our
>CsCl solution.  Obviously DS DNA floats in both near the middle of the
>tube.
>
>It made me ask myself, why KI and not CsCl?  Also, since we're pulling the
>lower band (we see only two, the lower is brighter) mightn't it be
>contaminated with some superhelical DNA along with the SS DNA?
>
>I really hate not knowing what's behind the reasoning for this protocol.
>
>Insights for why KI is used here and not CsCl would be appreciated, as
>would any favorite methods for purifying SS DNA away from DS DNA.  Some
>have suggested a low melt gel, but I'm hesitant, since we need a minimum of
>50 ug for each run.
>
>thanks 
>
>dennis

Instead of running gradients, why don't you treat the lysate with DNAse,
which will not get into the phage heads, then PEG ppt your phage (twice
if you want to be paranoid) phenol extract and etoh ppt. Should be
free of ds DNA.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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