mtDNA invertebrates universal primers

Black, Michael BlackM at dfo-mpo.gc.ca
Thu Jan 29 12:16:23 EST 1998


HaroldB786 wrote:
> I take it you have searched GenBank??? Unfortunately, I have only
worked with
> RAPD's for crabs..... See if you can find a copy of "The Simple Fools
Guide to
> PCR" by Bailey Kessing etal of University of Hawaii..... they list
universal
> primers.... including 16S.... if you can't find it..... drop me a line
and I'll
> transcribe them. 
> 
> But do your homework and find the papers..
> 
> Good luck,
> 
> harold berninghausen


Actually, in Eric's defense, yes, he has done his homework, or more
correctly I did it for him. 

His situation is this, in an attempt to try whole mitochondrion
amplification from crabs, I used the available Brachyuran GenBank
entries to design long PCR primers (working from the Nelson et al. paper
in Molecular Ecology, 1996, vol.5:807-810) in the 16S gene. So far,
after trying a whole variety of things he is only able to get a smear
for a product (even when using purified mtDNA). Some people from this
group suggested he actually test each primer with a small fragment amp
as long primers (these are both over 30bp) are difficult to synthesize
and the problem may actually be bad lots of primer.

One of the "universal" 16s primers will work quite nicely to test one of
my long primers, but we need something in the extreme 3' end of 16S to
test the other long primer, and I could not find any reference to a
"universal" primer that was downstream of my long one. Alternatively, if
12S is in the right place in a Brachyuran crab, we could use one of the
published 12S universal primers. Since I did not have a reference for
crustacean mtDNA gene order, I suggested he post to this group to see if
someone readily had such information. If they do, we would love to hear
from them.

Thanks, Michael Black

P.S. to Harold Berninghausen. My copy of "The Simple Fool's Guide to
PCR" is designated version 2.0 (August 5, 1991), compiled by Palumbi,
Martin, Romano, McMillan, Stice and Grabowski. Were you referring to a
updated version? If so, is it available anywhere? That little manual is
one of the first things I always gave my work study students and lab
interns.


--
Dr. Michael B. Black
Canadian Government Laboratory Visiting Fellow
Department of Fisheries and Oceans, Canada
Maurice Lamontagne Institute
850 Route de la Mer / PO Box 1000
Mont-Joli, Quebec
G5H 3Z4
voice: (418)775-0587
FAX: (418)775-0740
e-mail: blackm at dfo-mpo.gc.ca




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