Problems amplifying MtDNA

[Janet Williams]

Newsgroups: bionet.molbio.methds-reagnts
Subject: Problems amplifying MtDNA
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Organization: Oregon State University, Corvallis, Oregon
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I am amplifying four fragments of fish MtDNA.  I have had no problems
doing this until recently.  I can amplify three fragments of my
isolated fish MtDNA:  ND-1 (~2 kb), ND-2 (~1.2 kb) and ND-5,6 (~2.3 kb).
I can no longer amplify the D-loop fragment (~1.5 kb) consistently from
the same DNA.  But I can amplify two sub fragments of D-loop, using
the same two outer primers and a couple of internal primers.  One 
fragment is ~900 bp and the other ~600 bp.  I get very good yields 
amplifying these sub fragments.

I am using an Ericomp twin unit with the 96 well format and the 
heated top lid.  We amplify 190 samples at a time using the Robbins
Scientific thin wall PCR cycle plates.  I use a program that consists
of 5 mins at 95 (1 cycle) and 32 cycles of 45 sec at 95, 30 sec at
50 and 2 min 30 sec at 70; followed by a 5 min soak at 70.  The reagents
are the same for all PCR reactions, except the primers.

I would appreciate any advice anyone can give me.  I have about 
exhausted all the possibilities I can think of, including boiling
the primers and then putting them on ice.  I would especially appreciate
hearing from people experienced with using vertebrate MtDNA.

Janet Williams Hanus
williamj at bcc.orst.edu 


-- 
Janet Williams Hanus			Voice Phone: 503-737-4531
Department of Fisheries & Wildlife	FAX:	     503-737-3590
Oregon State University			e-mail:	     williamj at bcc.orst.edu
Corvallis, Oregon 97330-3803