How Much PCR product for sequencing?

Jean Renaud jrenaud at rsvs.ulaval.ca
Thu Apr 18 15:01:26 EST 1996


The best rule that I found is 5-10ng per 100bp,except for PCR products with
high GC content or secondary structures where I  use twice this quantity.
This rule give consistent results with well purified PCR products.
                Jean R.

Dear Autosequencers,
>
>We are in a bit of a dilemma - Does anyone have a good "rule of thumb"
>for the amounts of PCR product required for sequencing, per 100bp of
>product length? We are using Perkin Elmer Taq FS labelled terminator
>chemistry. We have found a lot of the manuals were written for
>ordinary Amplitaq. The PE kit instruction manual suggests different
>amounts from their Comparative PCR sequencing booklet for the same
>enzyme. Our users that are getting good direct sequencing results are
>not confident of the accuracy of their estimations.  Confusing, hey?
>--
>______________________________________________________________________
>
>Jennifer Cassady
>Manager
>DNA Sequence and Analysis Facility
>The University of Queensland
>St. Lucia, Qld. 4072
>AUSTRALIA.
>
>
>Ph:  +61-7 3365 4601
>Fax: +61-7 3365 4388
>email : cassady at biosci.uq.oz.au

Jean Renaud
Recherche en Sciences de la Vie et de la Sant=E9
Pav. Charles-Eugene Marchand
Universit=E9 Laval
Qu=E9bec,(Qu=E9.),Canada
G1K 7P4
t=E9l.:418-656-2552
fax.:418-656-7176
Internet:jrenaud at rsvs.ulaval.ca





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