Salt Gradient Sequencing gels

[Klaus.Matthaei at anu.edu.au]

>Does anyone have experience with salt gradient gels? I am trying one for
>the first time today and would welcome any advise. Specifically I'm wondering
>when to add the NaOAC if I am doing 2 loadings.
>The procedure I am using says to use 1XTBE in the bottom chamber, 0.5X TBE
>in the upper chamber and add 3M NaOAC to the lower chamber about 1 hour
>after loading.
>While I'm posting...
>I've had terrible problems with my sequencing gels leaking from the bottom
>while pouring. I seal the bottom with brown 3M mailing tape. Anyone have
>any hints for easy gel pouring?
>
>Thanks,
>Ethan Strauss                              IO00865 at maine.maine.edu
>Dept. Plant Biology & Pathology            208 Maple St. Apt#1
>University of Maine at Orono 04469         Bangor ME, 04401
>207-581-2994                               207-947-6017

Hi Ethan
I have been running gradient sequencing gels for many years.  I add a 1/3rd
volume of 1M NaOAC to the bottom buffer tank at the time of loading.  With
this method it does not help much to have two loadings but if you do then
add the NaOAC at the time of the second loading.

Cheers, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If you don't do: you rust"
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