Spotty microsatellite sequencing gels:HELP (was: bind silane and sequencing)

craig.primmer at bmc.uu.se craig.primmer at bmc.uu.se
Mon Aug 19 07:04:09 EST 1996


Hi everybody,

A sort of related problem from the bind silane topic of last week:

For a few months now we have had a problem with spots appearing on the films 
of our  microsatellite gels which seems to have been accompanied by the bands 
being more blurry than previously. We use a sequencing gel system which binds 
the gel to one plate (using Pharmacia bind silane) and repels from the other 
(using Rain-X, a windscreen cleaning product). This system has worked fine for 
over 3 years until just after Easter. We have since done a long series of 
experiments substituting one thing at a time from the PCR reactions and have 
bought new gel mix (new batch too), binding silane and repel. 

The dots appear to be in the gel as they only apear  in lanes where samples 
(32P) have been run, the more primer that has been used, the larger the dots. 
There also seems to be more dots directly around the well of the lanes. Also, 
the fragments appear to run through the dots and become streaky which makes us 
think that there are particles in the gel, but new gel mix does not solve the 
problem.We are at the point of considering that the composition of the binding 
or repel may have changed (ie- we are clutching at straws)

We have eliminated the PCR as the problem as we have run our samples on a gel 
in another lab and there were no dots

There is a pint of Guinness (or 2) in it for anyone that can solve this

Craig



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