Protocol

1. Dissolve HS Taq polymerase (Component #1) in Polymerase reconstitution buffer (Component #2). For this, add the whole volume of the Component #2 vial into the Component #1 vial. Resuspend carefully and wait until completely dissolved (~5 minutes).
2. Add 20 µL dsGreen for Real-Time PCR, 100x (Component #3) in the vial. Carefully mix the resulting blend, and spin down the solution using centrifuge.
   Important! After preparing and dsGreen dye adding, store the reaction mixture at −20 °С.
3. Prepare a reaction mixture according to the table in the proportion per (N+0.1N) reactions, where N is the required number of reactions. Add components in the given order. Carefully vortex the  resulting blend, and spin down the solution using centrifuge.
   Important! The volume of the reaction solution may vary depending on the specific application, but a volume of less than 10 µL is not recommended.
   Calculations for the total volume 20 µL per single reaction:
   The reaction components should be recalculated with the proportion below when using a different reaction volume.

   Component	Volume	Note
   2х Reaction mix	10 µL	—
   Forward primer	0.5–1.5 µL 10 µM solution	5–15 pmol/reaction (final concentration 250–750 nM)
   Reverse primer	0.5–1.5 µL 10 µM solution
   Deionized water	Is added to bring to the reaction volume of 20 µL
   DNA	2–9 µL (cDNA, 50–100 ng genomic DNA, 1–100 pg plasmid DNA)	Is added separately in each vial (see step 4)
   The total volume of reaction	20 µL

4. Add the final volume of the reaction mixture in separate PCR tubes without taking into account the volume of the DNA sample. Add DNA samples to each PCR tube, and spin down the solution using centrifuge.
   Important! To obtain reliable and reproducible data, run the PCR reaction at least two times for each DNA sample.

Amplification program

Use the standard method to calculate the primers' melting point (Tm) using Nearest-Neighbor algorithm (SantaLucia J. Jr., 1998). The primer annealing temperature is determined according to the equation: Ta=Tm–5 °C.

If the annealing temperature of the primers ≥60 °C:

Stage	Temperature	Time	Number of cycles
HS Taq polymerase activation	95 °C	5 minutes	1
Denaturation	95 °C	10 seconds	40
Primers annealing combined with elongation (Fluorescence detection should be performed at this stage)	60–72 °C	30–60 seconds

If the annealing temperature of the primers <60 °C:

Stage	Temperature	Time	Number of cycles
HS Taq polymerase activation	95 °C	5 minutes	1
Denaturation	95 °C	10 seconds	40
Primers annealing (Fluorescence detection should be performed at this stage)	55–59 °С	10–15 seconds
Elongation	72 °C	15–30 seconds

Important! After amplification, we recommend performing amplicon melting in a range from 60 up to 95 °С to ensure the absence of nonspecific amplification.

Related kits

dsGreen Lyophilized qPCR kit

Lyophilized, ready-to-use, high performance qPCR kit with hot start Taq polymerase and dsGreen fluorescent dye. All of the active components are freeze-dried allowing to be shipped at room temperature.

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