I am posting this protocol rather than reply individually to the
multiple requests I received.
Modified PCR protocol for yeast GenePairs
Primary amplification, genomic DNA >> amplified ORF
Per 50 ul rxn:
5 ul 10x thermophilic DNA Polymerase Reaction Buffer, MgCl2-Free (Promega)
(10x buffer =500mM KCl, 100mM Tris-HCl (pH 9.0 at 25°C),
1.0% Triton® X-100)
3.5 mM MgCl2
400 uM each dNTP
20 ng yeast genomic DNA
100 pmol each primer.
93oC-2 min
--------
30 cycles of:
93oC-30 sec
55oC-30 sec
72oC-150 sec
--------
72oC-120 sec
Secondary amplification, amplified ORF >> reamplified ORF is the same
as the primary reaction, with the following exceptions:
0.1 ng amplified ORF (cleaned up by ethanol precipitation) instead of
genomic DNA
50 pmol universal primer pair instead of ORF-specific primers (see
www.resgen.com for sequences)
annealing temperature is 60oC.
We have had good results for most ORFs on chromosome III that are 3
kb or less. Yields fall off with larger ORFs so we split larger ORFs
with our own primers. Occasional low yields (even some "no" yields)
on primary amplification are almost always compensated for during
secondary amplification. Most primary amplification failures are
corrected upon resynthesis of oligos by Research Genetics; a few ORFs
could only be amplified using primers further into the ORF (possibly
due to a strain sequence polymorphism or database error?). Two other
tips: we make our own yeast DNA, and we had the universal primers
synthesized, using the sequences supplied by Research Genetics.
Obligate disclaimer: Mention of any company is for informational
purposes and does not imply endorsement of their products
Nonobligate disclaimer: Although we use this protocol routinely, we
have not done exhaustive trouble-shooting--i.e. you're on your own
now.
Francis--could you port this over to the yeast listserve and save me
the trouble?
Michael Lichten
lichten at helix.nih.gov
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