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 =
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=
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

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