dna diffusion in agarose

Gary K. gtk10583 at glaxo.com
Wed Feb 21 09:15:02 EST 1996

In article <4gamor$682 at news.bu.edu>, rkoedood at bio.bu.edu (Riekeltje
Koedood) wrote:

> Ralph Schuster (gka1 at d2.hrz.uni-giessen.de) wrote:
> : 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 ?...

> Do you have EtBr in the gel or the buffer or both? May be a staining
> problem?
> Why do you want to run the gel overnight? 1-2 hours at 50-100 V should
> be sufficient to separate the bands.
> Marieke Koedood (rkoedood at bu.edu)

300 ug is a lot to load in one wide well.  EtBr migrates in the opposite
direction as DNA, the two smaller bands will "suck up" all the dye and
leave a wake behind with no dye.  The larger fragment will have no
exposure to the dye.  The recomendation here is to stain the gel
post-electrophoresis instead of running the gel with dye.

Gary K.

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