Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration.
The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.
MacPherson
J M
JM
Health of Animals Laboratory, Agriculture Canada, Saskatoon, Saskatchewan.
Eckstein
P E
PE
Scoles
G J
GJ
Gajadhar
A A
AA
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D014122:Toxoplasma / Q000235:genetics
D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S