Dna Fingerprinting Data for Use in Systematic , Ecological and Evolutionary Studies

The use of PCR based fingerprinting using short primers is very sensitive to variation in procedures. Strict laboratory procedures, exceeding general molecular biology standards, must be followed. Many laboratories have experienced difficulties in obtaining reproducible banding. Using a stock solution greatly minimizes pipetting small quantities, but the stability of a Taq stock is not well known. An investigation of the stability of a RAPDs stock (Taq, MgCl2, 10X buffer, dNTPs) revealed the stock to be very stable (for 4 d, and for 60 d at 22°C). Interim storage of DNA at 4°C was found to be a significant source of variability in banding. Variability due to different amounts of polysaccharide inhibitors in the DNA is a significant source of variation in banding. Experiments show that diluting the DNA to about 0.25 ng/rxn (15 μl) dilutes the effects of inhibitors, yielding stable banding. Thermocycler temperature variation between runs can be a problem and methods for monitoring are presented. Other sources of variability are discussed and remedies suggested.