User's Manual

15
We recommend running a melting curve analysis with the template DNA, dPCR primers, and Samplix reagents
to check for the presence of alternative amplicons and primer-dimers. Consider running a temperature
gradient to determine the optimal annealing temperature.
The supplied qPCR dye (20x) ● maximum values for excitation and emission are approximately 497 nm and
520 nm respectively (SYBR
TM
green settings). Make sure these values are selected in your qPCR instrument.
Calculate the PCR efficiency using the Ct values as input with the formula: (10^(-1/slope)-1) *100
Make sure that your designed primer pairs have an efficiency between 90-110 % and that the suggested DNA
input amount yields a Ct value below 30.
Fig. 2.1. Calculate PCR efficiency with at least three different concentrations of input DNA using the Samplix
Primer test PCR kit (Cat. No. RE10200) and your designed primers. Left: Amplification plot of three DNA
concentrations. Right: Standard curve and calculations of PCR efficiency.