377 base spacing problems

Paul Rasmussen paul at ib3.gmu.edu
Thu Jun 10 12:17:59 EST 1999

Frazer Murray wrote:

> Has anyone had problems with the base-spacing when analysing their 96 lane
> sequencing gels on the PE 377? I am running 48 lane gels fine with spacings
> about 10 or 11 but when running a 96 lane with the same conditions the
> spacings are given as defaults of red 9 and the sequences are unusable. It
> is as if the gel is running too slow. Do I need to increase the voltage
> that the gel is run at when running 96 lanes? At the moment it is at 1650V.
> I am using millipore collumns with G75 sephadex to clean up my termination
> reactions (big dye). Some people have suggested there is salt passing
> through into my sample but the gel image lookes fine and straight except
> for the very last few bases where it pinches in. I believe that salt would
> result in a more dramatic pinching.
> Any ideas would be most welcome
> --
> Fraze Murray
> Roslin Institute

I guess the first question is what is the WTR distance of your plates and what
Module are you using?  Assuming your WTR distance is 48cm the 48E1200 module
(for Bigdyes) has a default voltage of 2400 volts and should probably not be
changed significantly.  Similarly, the  36E1200 has 1680 volts and the 36E2400
has 3000 volts.   If the settings you are using are in line with these, I
would recommend running aliquots of the same exact samples on each the 96 and
the 48, rather than depending on separate samples under the same conditions.
This will eliminate the possibility of it being a salt related problem.  Good
luck and feel free to email if you have further questions.

PS  Now that you're doing 96 lane RXNs have you looked at the Millipore
cleanup PLATES?  They're dirt cheap (relative to other 96 well cleanup kits),
and we find, work quite well.

Paul Rasmussen
Research Associate
Institute for Biosciences, Bioinformatics, and Biotechnology
George Mason University, Manassas VA
Phone: (703) 993-8463     Fax: (703) 993-8460
paul at ib3.gmu.edu              www.ib3.gmu.edu

"Whenever I watch TV and see those poor starving kids all over the world, I
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