Neurospora crassa PCR

Matthew Sachs msachs at ADMIN.OGI.EDU
Sun Jul 2 13:34:20 EST 1995


>I am having a difficult time PCRing N. crassa and I was wondering if
>anyone could direct me to a paper that could guide me on optimal
>conditions i.e. buffer, template and primer concentrations, enzyme
>amount, temperatures and cycle number.

This is a protocol Michael Freitag and I have used successfully for
amplification of a 662 nt reporter DNA fragment from plasmids and from a
single copy integrant in the Neurospora genome using an Ericomp cycler:

In a typical reaction (50 =B5l), 5 ng plasmid DNA or 100 ng genomic DNA
template, 0.5 =B5M primers, 400 =B5M of each dNTP, 1X VentPolymerase buffer =
(10
mM KCl, 20 mM Tris-HCl [pH 8.8], 10 mM [NH4]2SO4, 0.1% Triton X-100; New
England Biolabs, Beverly, MA) and VentPolymerase (1 u) were overlaid with
25 =B5l of light mineral oil (Aldrich, St. Louis, MO) and cycled 35 times
through the following temperature profile: 90 s at 94=B0C, 30 s at 50=B0C,=
 60 s
at 72=B0C.  The last extension step was carried out for 5 min.  Reactions
were cooled on ice and extracted with 60 =B5l chloroform, vortexed and phase=
s
separated by centrifugation for 5 min at 16,000 xg.  One-tenth of the
reactions were run on an analytical 0.8% agarose gel.  Reactions that
yielded discrete bands (662 nt) were directly digested with EcoRI [the
fragment had internal EcoRI sites] and used for subcloning into pBS SKII.


-----------------------------------------------------------
Matthew Sachs
Department of Chemistry, Biochemistry and Molecular Biology
Oregon Graduate Institute of Science and Technology
20000 NW Walker Road
P.O. Box 91000
Portland, OR  97291-1000
503 690-1487 Phone
503 690-1464 Fax
msachs at admin.ogi.edu





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