IUBio

How Much PCR product for sequencing?

Vahe Bedian dnaseq at mail.med.upenn.edu
Thu Apr 18 09:48:53 EST 1996


>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?
>--
>______________________________________________________________________
Jenny,

We routinely use 10 ng/100bp with the same chemistry. Gives a strong
signal. Could use less if you needed to.

We find that salts and alcohol from the purification columns can give
variable inhibition of the sequencing reactions. A microcon 100 cleanup or
a precipitation makes results consistent. Best way to quantitate is
probably on a gel, although we have a 50 ul microcuvette that allows us to
scan and check for purity (both salts and organics give high absorbance at
200 nm).

Vahe Bedian
DNA Sequencing Facility
Univ of Penn - Genetics





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