377 base spacing problems

daemon at net.bio.net daemon at net.bio.net
Mon Jun 28 10:16:30 EST 1999


On 8 Jun 1999 13:54:26 -0700, Frazer Murray
<Frazer.Murray at bbsrc.ac.uk> 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

Sequencing parameters are as follows: Electrophoresis voltage 2700V,
Electrophoresis current 60mA, Electrophoresis power200W, gel
temperature 51°C, laser power 40mW, CCG 4, collection time of 3.5 hr,
using 5% Long RangerÔ preMix gel solution (FMC BioProducts, 50815).
  
How are you perfoming your pre-runs for loading odd and even samples?
Ours are as follows:
Load lower buffer chamber with 1 X buffer, load the upper buffer
chamber with 540 ml distilled water.  Load even samples first and
perform a 2 minute prerun.  Load odds and perform an additional 2
minute prerun.  After preruns are completed add 60 ml 10 X buffer to
the upper chamber and complete your sequencing run.  This is
specifically for 96 lanes.

Hope this works well for you.

Labrat29
Clemson University
Dept. Micro. & Mol. Med.
SC
"Our university focuses on athletics."




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