Where in gel is DNA?
dspinella at chugaibio.com
Wed Aug 27 09:39:36 EST 1997
Dr. Volker Blaschke writes:
> I am trying (more or less successfully) to extract PCR products from
> agarose gels. So far, I have been cutting out the slices from the lanes,
> thus having a lot of gel mass to start with.
> I have noticed that when I had the slices "flat" on the
> transilluminator, that the fluorescence is mainly located on the UPPER
> part of the gel and not evenly distributed over the whole depth of the
> slice, and this is also true when having EtBr-stained the gel for an
> hour or so, so that for sure the whole slice is evenly soaked.
> So, should I still take the "whole" slice or just excise the small "top"
> Any input is highly appreciated...
I suspect that the reason your DNA is not distributed throughout the
entire thickness of your agarose gel slice is that your comb only
protrudes into the liquid agarose to a shallow depth (which of course
defines the depth of your wells). When you apply current, the DNA
migrates too quickly to diffuse deeper and so it just moves along the
top surface of your gel -- the same depth at which it started.
Alternatively, if you load your wells with the DNA in too small a volume
relative to the dimensions of the well, the DNA will also migrate in a
narrow band corresponding to the height of the solution you loaded. Yes,
you can simply cut away the excess agarose and use only the EtBr stained
portion to recover the DNA. If you find that you are "overloading" your
gels (your resolution is poor), try adjusting the comb depth when you're
pouring them so you get deeper wells and thus more surface area through
which the DNA can migrate. (But be sure that the comb does not touch the
bottom of your gel box, else the DNA will just leak out the bottom of
the wells). Hope that helps. -- D.G. Spinella
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