RE: Sequencing PCR Products onthe abi377XL

Miller, Mark J. (millerm@dc37a.nci.nih.gov)
Fri, 24 Jul 1998 09:17:41 -0400

Sequence Analysis scales the peaks in an attempt to optimize the readability of
the chromatograms. With short sequences like this, where only a small portion of
the entire scan may contain data, the program over compensates. It is necessary
to set the stop point in the Sample Manager to be at or just beyond the end of
the sequence (look at the raw data graph to determine this point). Rerun the
analysis with the shortened stop point and the peak heights should be in better
scale. Note: if you've put too much PCR product into your reaction you may
saturate the machine. Again check the raw data graph. If a lot of the data is
above 4000, you MAY have a problem. Dilute your sample 1:5 (1:10?) and run
again.

Mark Miller

-----Original Message-----
From: robert obrien [SMTP:rfob@email.msn.com]
Sent: Thursday, July 23, 1998 10:21 PM
To: Recipients of ABRF List
Subject: Sequencing PCR Products onthe abi377XL

I have recently begun sequencing 200bp pcr products on the 377XL and
have
been noticing the following abnormalities: Peaks have a flat top
appearance
to them and there are peaks under larger ones. Is it customary to get a
lot
of N calls? What% is acceptable to get?

Any Suggestions will be greatly appreciated

Thank You

Robert O'Brien