When Low Melt bands are contaminated???
BOTJLH at LURE.LATROBE.EDU.AU
Sun Jan 8 03:17:11 EST 1995
In <pemanuel-0501950943340001 at beaker.ab.umd.edu> pemanuel at umabnet.ab.umd.edu writes:
> I recently ran a 1% low melt gel isolation of a 700 bp PCR fragment. When
> I cut out the band and isolated it with GENECLEAN I found that a
> contaminating band of 1400 bp had gotten in there somehow. A friend said
> that can happen if you pverload the gel. The top band piggybacks on the
> bottom because the gel is overloaded. Load less was the recommendation.
> If I load less then I must cut out a bigger band. If you know anything
> about GENECLEAN you know that cutting out a bigger band spreads the
> isolations out over several tubes and can really lower your yield.
> So the questions are:
> 1. Why is there a contaminant in there at all?
> 2. How can I avoid it? Or how much DNA can I load in a single well?
> 3. Would a lower percentage gel or running the gel a real long time avoid
> this problem?
> Thanks for your suggestions on this one.
Try heating the sample to 65C for a few minutes before loading. I have heard
that this can be used to stop piggy-backing of the 4.3 kb band to the 6.5 kb in
the lambda EcoR1 mwt markers.
Maybe it will work.
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