SS Phagemid gradients, KI or CsCl?

Dennis J. Templeton djt2 at po.CWRU.Edu
Tue Jul 21 10:40:49 EST 1992


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



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