RAPD patterns of macronuclear genomes in P. caudatum

Cycling parameters employed for producing RAPD

by Y. Tsukii

Macronuclear DNAs were amplified with random 10 (or 8)-mer primers in a thermal cycler (Zymoreactor, ATTO Inc.). Amplification reaction volumes were 30 ul, each containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 300 uM each of dATP, dCTP, dGTP, and TTP, 0.01% Triton X-100, 0.6 ug primer, 5 ng template macronuclear DNA and 0.5 U Taq polymerase (Perkins Elmer-Cetus). Thirty-one primers as shown in table 1 were synthesized by TAKARA Inc. and InterTech Inc.

PCR was essentially according to the method described in Williams et al. (1990) except cycling parameters. Prior to investigation, optimal cycling parameters were examined (see below) and found 0.5 min at 90 C for denaturation, 1.0 min at 30 C for annealing and 4.0 min at 50 C for extension (30 cycles) (Prog. 7) . These parameters were employed throughout this study.

Primer IT-01; Template Nn4a macronuclear DNA; 0.8% Agarose gels were stained with azure C;
Marker lambda DNA Sty I digests;