multiplying bands in gel purification

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Thu Feb 20 07:43:53 EST 1997


   Did you have the 4.4kb band in the original gel?  If you did, you 
have just rediscovered that electrophoresis is qualitative, not
quantitative.  It is not something which is talked about, but I see it
quite often.  The closer the bands, the worse the problem.  I
originally found this out (the hard way) when trying to send an insert 
from one vector to another, and mostly got the first vector in my
transformations.  Now I mostly see it when I am purifying inserts for
random primer labeling.  I purify about 20ug of cut plasmid, cut out
the insert band (which is usually well separated from the plasmid
band), extract, re run the excised band, and voila, there is some more
of the vector band that shows up.  I have to double purify because the
residual vector labels up my RNA marker lane too much, and can
sometimes obscure my signal.  It's a pain in the butt, but that's
life.

						Regards		SVEN


In article <330A4042.2552 at mail.med.cornell.edu>, jgluz at MAIL.MED.CORNELL.EDU ("John G. Luz") writes:
> Folks--I'm tearing my hair out.  I cut a nice clean tight band out of a
> gel, and purified it with the qiagen gel extraction kit.  But when I ran
> the gel-purified band out on a gel to check my yield, I got two bands,
> both about equimolar.  One band looked about the right size.  The other
> looked about 1kb smaller.  The band I gel-purified was 5.4 kb and cut
> with NdeI and Xho.  What's going on?--John



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