Robert,
The flat-top peaks with additional colors are A's, right? Especially
noticible in the early part of the run? We see exactly these symptoms
when the template concentration is too high. Note that the signal levels
may not appear inordinately high, however. I suggest you dilute your
templates; start with a factor of 4 or so, and try a range up and down
from there to determine the ideal.
PCR reactions should sequence quite well, with few or no N's until the
final pile-up at the end of the template (where you may lose a base or
two). Certainly errors should be no more frequent than with any other
template type.
Bob Lyons
University of Michigan
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Robert Lyons, Ph.D.
Director, DNA Sequencing Core
University of Michigan
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