Essential components for reliable RT-PCR are included in each single LEADSPHERE® One-Step RT-PCR Master Mix, which saves time for reaction set-up and reduces the risk of contamination and pipetting errors. In addition, the stabilized format facilitates shipment and storage without cooling. LEADSPHERE™ One-Step RT-PCR Master Mix includes reverse transcriptase, DNA polymerase, high-quality dNTPs, and optimized PCR buffer. This product is suitable for probe-based detection and endpoint DNA amplification. The reaction can be set up by reconstituting the LEADSPHERE™ One-Step RT-PCR Master Mix by adding PCR-grade water along with the RNA template, primers, and probe to a total volume of 20 μL.
- Primer/Probe concentration
Final concentrations of 400 nM (each primer) are suitable for most reactions. To obtain optimal condition, primer concentration can be titrated between 0.2-1 μM.
A final concentration of 200 nM (probe) is suitable for most reactions. To obtain optimal condition, probe concentration can be titrated between 0.1-0.3 μM.
- Annealing/Extension optimization
To obtain optimal condition, annealing/extension temperature can be adjusted between 55°C-65°C, annealing/extension time can be extended up to 60 sec.
- Target length
Appropriate amplicon length should be arranged between 80-200 bp