dna diffusion in agarose

Ralph Schuster gka1 at d2.hrz.uni-giessen.de
Fri Feb 16 09:01:38 EST 1996

Hi everybody,

I'm having a somewhat unusual but sometimes reproducible problem.
I'm trying to obtain a large amount of insert DNA by first cutting
my vector with two enzymes, one that cuts the vectorpart in two bits
and one that cleaves my insert out of the vektor. I use about
300 µg DNA, and the digestion is no problem at all. After that,
I put the whole solution (about 200 µl) on a 0.7 % agarose gel
to separate my insert (3kb) from the vectorparts (1.2 and 1.6 kb),
and let the gel run overnight at 25 volt. Normally, one would expect
three seperate bands on the gel, but what happens is that the
1.2 and 1.6 bands are just fine, clearly visible and all, but the
3 kb band seems to have more or less vanished in the process, there
is still a small amount visible,  but the rest is gone. The question
remains: where does it go ?? Is there a kind of party in my
eletrophoresis that only 3 kb bands can attend, but smaller bands
are not allowed ? It just seems as if the 3kb part is somehow
diffused over the gel, but I have no explanation as to why.
Did anyone ever have similar problems, or can give me a hint
on how to avoid this ? I never had problems like this before, and
I already tried different electrophoresis apparatures, different
agarose and different volts, all to no avail. The puzzeling
thing is, in the middle of all this it worked once, but I don't
know why, and I have three different plasmids which to put through
this whole procedure.
Help, anyone ??

Ralph Schuster
Med. Virology
Ralph.Schuster at viro.med.uni-giessen.de

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