whole mtDNA PCR

Black, Michael BlackM at dfo-mpo.gc.ca
Thu Nov 27 11:10:07 EST 1997


Hello everyone,
   We're going to try and amplify the whole mitochondrial DNA molecule
(less some 300-350 bp) from a species of crab. I used the available
brachyuran 16S fragments in GenBank to design two primers, a 33-mer (Tm
=  73.5C, 30 sites appear to be "universal brachyuran" sites) and a
27-mer (Tm = 73.2C, 24 sites appear to be "universal brachyuran" sites).
We will be using purified mtDNA (mitochondrion isolation followed by
lysis and a standard phenol/chloroform DNA extraction).
   If anyone has any experience trying this sort of thing, I'd
appreciate any hints or suggestions. Any thoughts on reaction conditions
would be particularly appreciated. Given the Tm's, we were thinking of
using a two-step cycle? Thanks in advance.

--
Dr. Michael B. Black
Department of Fisheries and Oceans, Canada
Maurice Lamontagne Institute
e-mail: blackm at dfo-mpo.gc.ca




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