Problems amplifying MtDNA
williamj at ava.bcc.orst.edu
Wed Jan 25 20:17:15 EST 1995
Subject: Problems amplifying MtDNA
Organization: Oregon State University, Corvallis, Oregon
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
More information about the Methods