Strange problem with agarose gel electrophoresis

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon Apr 16 19:06:39 EST 2001


Hmmmm ... happens closer to the top and if that is "RED" then you are
running the gel bacwards. Correct me if I understood it wrong ... by "top",
do you mean the "starting" end of the gel? That end should not be "Red" ;-)

DNA migrates from "Black" to "Red" ... I have no idea why! Somebody long
long time ago decided those colors for everybody else, and since then
nucleic acids have been migrating from the "Black" electrode to the "Red"
one -- provided a potential difference exists in between.

If this does not solve the problem, I may suggest preparing the TBE stock
(5x or 10x, whatever you have) fresh. I don't use TBE for routine DNA gels
for its notoreity in going "bad" (precipitating) within a few weeks.




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.
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu

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