DNA was amplified from single extractions to minimize variation in DNA quality and concentration. Negative controls were run to prevent scoring artifact bands.
Screening and scoring procedure Preliminary screening for variable RAPD markers was conducted using one sample each from the Quinnault district of Olympic National Forest, Washington; Cle Elum district of the Wenatchee National Forest, Washington; and Mapleton district of Siuslaw National Forest, Oregon. These samples were screened for variability with 400 standard RAPD 10-mer primers (sets 100/2, 100/3, 100/4, 100/5) from the Oligonucleotide Synthesis Laboratory, University of British Columbia. Each variable band was scored as present (1) or absent (0) and was considered to represent the phenotype of a distinct locus. We took a conservative approach to scoring by choosing only distinct, intense, and highly reproducible bands. Negative controls were run with each primer to insure that scorable fragments were not artifacts.
DNA was obtained by a standard phenol/chloroform extraction as previously reported (Haig et al. 1994). Briefly, 10ul of blood was digested in 400ul of extraction buffer (50mM Tris pH 8.0, 10mM EDTA pH 8.0, 200mM NaCl, 2% SDS) with Proteinase K (20mg/ml) added to a final concentration of 600 µg/ml. Samples were then vortexed and incubated overnight (~18hrs) at 50° C. If blood clots were not fully dispersed, a second aliquot of Proteinase K was added and samples were incubated an additional 2 hours. Samples were extracted with equal volumes of phenol (saturated with 10mM Tris pH 8.0) and then chloroform/isoamyl alcohol (25:1). DNA was precipitated by adding a 1/10 volume of 3M sodium acetate and two volumes of cold 95% ethanol and pelleted at approximately 15,000 x g for 20 minutes in a microcentrifuge. Pellets were washed once with 70% ethanol, dried under vacuum and resuspended in 30ul TE (10mM Tris pH 8.0, 0.1mM EDTA pH 8.0). DNA concentration of samples was quantified with a Hoefer TKO 100 fluorometer and working solutions of 0.5 ng/ul were prepared for experimental use.
Varying DNA quality and concentration can alter RAPD results (Perez et al. 1998). To minimize this problem, we used 2 ng total DNA for PCR reactions from a single extraction (pool) of DNA for each sample. DNA was run out on agarose gels to insure it was not degraded. Replicates of samples were amplified in side by side PCR reactions and in multiple PCR runs to verify profile reproducibility for all fragments scored.
RAPD PCR procedure Screening of primers and scoring runs were produced on a MJ Research model PTC-100 programmable thermal controller, using optimized conditions outlined in Aagaard et al. (1995). A total reaction volume of 25ul was used with the following concentrations: 50mM KCl; 10mM Tris-HCl at pH 9.0; 0.1% Triton X-100; 1.8mM MgCl2; 100µM for each of the dNTPs; 0.2uM primer; 2 ng template DNA; and 1 unit of Taq DNA Polymerase (Promega). The following parameters were used for the amplifications: 3 min. denaturation at 93° C,followed by 45 cycles of 1 min. at 93° C, annealing at 37° C for 1 min., and elongation at 72° C for 2 min. A final 10 min. elongation at 72° C followed the last cycle. Amplification products were electrophoresed in 2.0% agarose gels (GibcoBRL; Ultrapure) at 100V for 4 hours in TBE buffer (90mM Tris base, 90mM Boric acid, 2mM EDTA, ph 8.0). A 1 Kb DNA ladder (GibcoBRL) was used as a molecular size standard. Gels were stained in 1µg/ml ethidium bromide for 30 min. and destained in distilled H2O for 2 hours.
Screening and scoring procedure Preliminary screening for variable RAPD markers was conducted using one sample each from the Quinnault district of Olympic National Forest, Washington; Cle Elum district of the Wenatchee National Forest, Washington; and Mapleton district of Siuslaw National Forest, Oregon. These samples were screened for variability with 400 standard RAPD 10-mer primers (sets 100/2, 100/3, 100/4, 100/5) from the Oligonucleotide Synthesis Laboratory, University of British Columbia. Each variable band was scored as present (1) or absent (0) and was considered to represent the phenotype of a distinct locus. We took a conservative approach to scoring by choosing only distinct, intense, and highly reproducible bands. Negative controls were run with each primer to insure that scorable fragments were not artifacts.