long PCR

[Aawara Chowdhury]

In <mailman.981.1178324769.5139.methods from net.bio.net>,
 Mandeep Sharma <mxs781 from psu.edu> wrote:

> I am trying to amplify a 10kb maize genomic DNA fragment using long PCR.
> I have already tried Pfu ultra fusion from promega and geneamp high
> fidelity PCR system from applied biosystems and they didn't work. If you
> have tried amplification of this size fragment and you got the
> amplification then can you please suggest which taq polymerase should I
> use and what will be the most suitable conditions for long PCR with that
> taq?

I strongly suggest you read the original "long & accurate PCR" paper
in PNAS by Wayne Barnes:
<http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8134376&query_hl=11&itool=pubmed_docsum>

In my lab, we mix Taq and Vent in a unit ratio of about 500:1 to amplify
fragments from human genomic DNA that have ranged from about 3 kb to 8 kb
using this mixture, and the reaction conditions described in the Barnes
paper.

Recently, a visiting scientist had to amplify a long portion of Leishmania
DNA, which is more GC-rich than the human genome.  The Taq/Vent mixture
failed, unless we added Betaine in a final concentration of 1 molar to
1.5 molar in the PCR.

AC
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