EffiStart™ 5X One-Step Probe RT-qPCR Master Mix is a one-step real-time reverse transcription-polymerase chain reaction (RT-qPCR) kit developed for cDNA synthesis and real-time PCR in the same tube. This product contains Hot Start Taq DNA Polymerase and is suitable for probe-based detection and formulated as a 5-fold premix. Reaction can be simply set up by adding the RNA template, primers, and probes. This master mix does not contain ROX reference dye; it offers great convenience and minimizes the risk of cross-contamination.
- 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.