Loading Long Ranger gels...

John W. Longshore Gene009 at uabdpo.dpo.uab.edu
Tue Nov 1 15:27:54 EST 1994

In article <395651$jag at eldborg.rhi.hi.is>, php at rhi.hi.is (Petur Henry
Petersen) wrote:

>         I was wondering... When running Long Ranger Sequencing gel (6%)
> I usually wait until the gel is around 45 degr and run it at about 45-50 degr
> (Celcius of course :) ). In the Long Ranger leaflet they say one has only
> to prerun the gels for 10 min. After 10 min prerun my gels are about 35 degr.
> Should I load at 35 or wait as I usually do? I am trying to get sharper
> bands and one of the factors I am concerned about is the heat of the gels..
>         Could anyone tell me what they do? I should mention that I am running
> sequenced PCR products and they show litle secondary structure and that I
> use biotinlabeled primers to get ssDNA for sequencing...
>         Petur Henry Petersen DeptmBiology U.Iceland

After 10 minutes, the temperature at the middle of the gel is 35 degrees,
but the temperature at the bottom and the top of the plates is
higher(Assuming the thermometer is in the middle of the plate).  I prerun
5% LR gels 10 minutes and have no problems.  I have prerun the same gels
20 minutes, but the quality seens to be the same.  Use a buffer gradient
to sharpen your bands.  AT Biochem used to recommend runnimg the gel in
0.6X TBE buffer with the gel containing 1.2X TBE.  This works well for me!

Hope this helps!

John W. Longshore
Laboratory of Medical Genetics
University of Alabama at Birmingham
gene009 at uabdpo.dpo.uab.edu

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