Real-time PCR buffer

The PCR buffer consisted of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 4.5 mM MgCl2, 0.05% Nonidet™ P-40, 0.05% Tween® 20, 0.03 % acetylated bovine serum albumin (BSA), and 200 μM each dATP, dCTP, dGTP, and 600 μM dUTP. Reactions were performed in glass capillaries with 15 μL of 1.33× master mixture and 5 μL of sample nucleic acids or standards. Each 20-μL reaction contained 2.0 U Platinum® Taq DNA polymerase (Invitrogen), 0.2 U heat-labile uracil-N-glycosylase (Roche Applied Science), and 0.0213 U ThermoScript™ reverse transcriptase (Invitrogen). PBGD primers were used at 0.8 μM, all other primers at 1 μM, BODIPY 630/650 and Cy5.5 probes at 0.2 μM, and 6-FAM probes at 0.1 μM. Master mixtures were prepared freshly from 10× PCR buffer, 5× oligonucleotide mixture, 50× nucleotide mixture, and enzymes. For convenient pipetting, ThermoScript reverse transcriptase was used at a 1:140 dilution in storage buffer. Standard reactions containing 10,000, 1000, 100, or 10 copies of each target were performed with each run.