DNA stuck in agarose gel wells
Harry J. Witchel
Harry.Witchel at bristol.ac.uk
Wed Jan 24 07:56:27 EST 1996
In article <boehnin1-2301961430170001 at joseph2.tjh.tju.edu>,
boehnin1 at jeflin.tju.edu} says...
>I have been experiencing problems with DNA being stuck in the wells.
>Although we do find that some of the sample comes out and is seen as a
>faint band but most of the remains in the well.The well is not
>contaminated with pieces of agarose. The wells are well formed too. I
>would appreciate any suggestions.
Hello Shaila --
You do not say how you know that it is DNA (rather than salt) which is
stuck in your lanes. You also do not say whether the DNA is large genomic DNA
or small plasmids. First of all, salt (eg NaOAc) will light up under EtBr in
the gel well. Salt can be reduced by a good washing in 70% EtOH after normal
EtOH precipitation. If it is plasmid which is stuck in the gel well, then you
have a contamination problem (salt, organics, something other than DNA).
If it is genomic DNA which is stuck in the gel well, then you probably
have a resolubilization problem. Genomic DNA is notoriously difficult to
redissolve once it has been precipitated; at the very least, you should allow
one hour in the fridge to dissolve in TE, and overnight is better. If you
have overdried the DNA or if it is still contaminated with certain kinds of
carbohydrates when it is precipitated (this may happen with plants, marine
invertebrates, and other gooey animals), the DNA will never go back into
solution, so you will have to find a new DNA preparation.
Sometimes it is difficult to get genomic DNA into the gel matrix. If
you think your DNA is getting stuck in the well as it is trying to enter the
gel, then you might try to run lower concentrations of gel (for genomic you
should be using 0.3-0.6%, but you will need good substrata and a cold room for
running these gels, as they fall apart very easily). You can also run the gel
much slower at the beginning 15 minutes (as the DNA enters the gel matrix).
For high MW DNA (>25kb) I start the gel at 30V (2 V/cm), whereas I run most
gels at 70V (4-5 V/cm). Good luck, and if you have more specific questions:
Best wishes and you can e-mail me at
Harry.Witchel at Bristol.Ac.Uk
>Thomas Jefferson University
>E-mail at Bokkala1 at jeflin.tju.edu
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