ABI 48cm strech problem

Bob Chadwick chadwirb at ccmail.apldbio.com
Thu Feb 9 11:33:01 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 your spacing is -12, your gel is running too fast (most common) or
too slow (not very common).  Since you say that your peaks overlap,
almost certainly your gels are running fast.  Make sure that your
10XTBE is good (i.e. freshly made at right concentration and no
precipitates). Also check out how old your acrylamide solution is. 
Once in solution acrylamide is stable for only a couple of weeks (or
less if it is kept in a clear container under bright lights or
dissolved in impure water).  I've also found that some gels made from
certain suppliers of acrylamide will run fast.  I personally use
IBI/Kodak acrylamide/bis (19:1) and my gels always run at the correct
speed.



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