ABI 48cm strech problem

John H McDonald mcdonald at strauss.udel.edu
Thu Feb 2 11:25:55 EST 1995


In article <9502021431.AA00258 at phage.cshl.org>,
Muhammad Lodhi <lodhi at CSHL.ORG> wrote:
>Lately we are having lots of trouble with our 48 cm strech.  We have tried
>running gels with Taq terminator and dye primer rxns on it and most of the
>sequences, even though look good on gel, are pretty bad.  Sequences are
>noisy, base spacing is -12 and peaks tend to overlap.  This is mostly but
>not entirely true for rxns with forward primers.  I am wondering if anyone
>has any idea how to correct it or what are we doing wrong.  Is there any
>problem with the sequencer matrix of filter set A.  Any help/advice will be
>appreciated.

If the Gel Image looks good, but the analyzed data are crappy and the base
spacing is -12, your gel probably ran either too fast or too slow.  The
version of the Analysis program that I have throws up its hands in
confusion at any peak spacing less than 9 or greater than 15 scans/peak
and uses a default value of 12.  Since peak spacing is an important
parameter in the algorithm, this leads to junky looking analyzed sequence. 
Check the EPT windows to see if your voltage is different now than it used
to be when things were working well, since higher voltage=faster running
gels=fewer scans/peak.  My guess is that either someone made up a new
batch of buffer at a slightly different ionic strength, or someone forgot
to filter out the Amberlite beads before adding buffer to the gel mix, or
someone has been messing with the settings on the machine. 

John H. McDonald
Department of Biology
University of Delaware



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