The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature. (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H2O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.