pCRII Topo sequences, pUC119 doesn't: why?

Nicolas Lemee Lemee at attglobal.net
Thu Feb 10 13:23:56 EST 2000

We had trouble sequencing a big insert in a pUC type vector, given all the
controls we made, we thought on a long shot that there might be some
conformation problems. Anyway, we linearized the plasmid, used that as a
template, it worked.

Biopolymer Facility wrote:

>         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
> _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
> 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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