Fw: Strange problem with agarose gel electrophoresis

Deanne Bell dbell at qnis.net
Tue Apr 17 12:09:56 EST 2001


My first instinct is to say that you are running your gel backwards also.

DNA has a slightly negative charge and thus migrates from the negative
"Black" electrode towards the positive "Red" electrode.  It sounds like
your samples are running right off the gel into the tank buffer.  You can
also get this problem by plugging the electrode wires into the power supply
backwards [personal experience ;-)].  I always check my gel after about 5
minutes of running to make sure everything is going all right.

I however disagree with Dr. Roychowdhury about the TBE buffer.  I keep a
10X Stock solution around for months without any ppt problems.

I am not experienced with your electrophoresis set up.
I get the impression that there are 2 sets of 48 wells;
if these on 2 different tiers, I would say that you are running the gels
backwards somehow;
if there is a distinction between the left and right half of the gel, I
would wonder if there is a short somewhere in your setup.

: 
: 
: At 05:02 PM 4/16/01 -0400, Arjun Sivasundar wrote:
: >I'm running PCR products on 3.5% Metaphor agarose gels (in 1X TBE) to
: >separate
: >small PCR products from microsatellite loci. The gels are the large 96
: >well type, run in
: >0.5X TBE buffer, at 400 volts with cooling. This lab has been running
: >gels this way for
: >a while now without problems. What I have started seeing is that the
: >products in the
: >one set of (48) wells rapidly start disappearing and in about 10 minutes
: >there is nothing there...no DNA, no loading buffer, no DNA ladder
: >either. The other 48 samples run just fine. This has happened both to
: >the top set of wells closer to the red (+) end as well as the set closer
: >to the black (-) end but not both at once (so far at least). I have
: >tried changing the running buffer, tried a new batch of agarose, cleaned
: >the electrodes etc. but it persists. If somebody can help it would save
: >me a lot of PCR products (and time). Thanks a lot.
: >
: >

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