We used cycle threshold (Ct) and melting temperature (Tm) values to discriminate amplified cDNA products from primer dimers (Fig. 1b, Additional file 1: Figure S3).
The results showed that fluorescence intensity of the hybrids decreased as the temperature increased in a target-dependent manner (Additional file 1: Figure S2, left panel), yielding different melting point (Tm) values for each target derived from the melting point (Additional file 1: Figure S2, right panel).
Melting curve detection to determine the melting temperature (Tm) values for the target sequences was set as follows: 1 min at 95°C, 2 min at 40°C, 75°C continuous and then cooling at 10 sec at 40°C.
Based on this mechanism, the melting temperature (Tm) values of reAuXyn11A and reAEXynM were graphically determined from the derivative melting curve of 57.6 and 91.6°C, respectively, which corresponded well to their temperature stability levels.
Melting temperature (Tm) values were obtained from the minimum of the first derivative of the melting curve.
The melting curves were normalized according to the methods in the literature (Arnórsdóttir et al. [2002]), and the melting temperature (Tm) values of the enzymes were estimated using a graphics software package, Delta graph.
After amplification, melting curve analysis at 65°C to 95°C was performed with a temperature transition rate of 0.1°C/second to determine the melting temperature (Tm) values for amplified PCR products.
The melting temperature (Tm) values for the synthesized polymers determined from DSC are higher than the PEG used (−38°C for PEG-200 and 49.52°C for PEG-2000, vavailableilable from Merck product data sheet).
A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (Tm) values of 73.85 °C (nheA), 87.01 °C (hblD), 78.66 °C (ces) and 82.19 °C (cytK1).
The melting temperature (Tm) values of SC crystallites in the blend (189 193 °C) were slightly higher than those of homo-crystallites in the neat P L-2H3MB) and P L-2H3MB) (163–178 °C).
