Mark,
How long are the amplification primers? I've found that longer PCR
primers (30mers and up) don't
necessarily make good sequencing primers. Try a nested sequencing
primer designed at a more optimal
length (18-24mer). If you are using "Big-Dye" chemistry the "T" blob is
probably unincorporated material
which according to PE can aggregate and migrate at a higher molecular
weight on your gel. If the
extension reaction is not efficient your usual terminator clean-up may
not handle the excess terminator load.
Ken Idler
DNA Sequencing Core Facility
Abbott Laboratories
ken.idler@abbott.com
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