pCRII Topo sequences, pUC119 doesn't: why?

Biopolymer Facility biopolymer at sc3101.med.buffalo.edu
Thu Feb 10 12:33:29 EST 2000



	Hello to everyone. I work in a DNA sequencing core facility and
have a customer who has what seems to me a peculiar problem. Hope someone
can help...
	Situation: starting DNA is pUC119 with approx. 9kb insert. PCR
primers are designed to amplify up sections of this insert (the sequence of
the insert is known) and then these subsequent fragments are cloned into
pCRII-Topo. To confirm success, the fragments within the Topo vector are
sequenced using the PCR primers and vector primers as sequencing primers.
All primers work well and give the expected sequences. Now the problem -
when attempting to use these same primers to sequence off the original
complete insert within the pUC119 vector, many of these primers give no
sequence, including the M13 vector primers. The primers that do work give
beautiful data, so I don't really suspect the quality of the pUC DNA. The
primers being used are from the same tubes as those used when sequencing
the Topo fragments so it's not a case of the incorrect primers being
provided.
	Am I missing something basic here? Does anyone have a possible
solution?

Thanks in advance...

Michelle Detwiler








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Biopolymer Facility
Roswell Park Cancer Institute
Buffalo, NY   14263
Phone: (716) 845-8032
Fax: (716) 845-7621
Email: biopolymer at sc3101.med.buffalo.edu
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