Description
The StableMax RT-qPCR Master Mix (with Dye, UDG) is a one-tube, probe-based RT-qPCR premix optimized for both single and multiplex RNA detection. Its unique buffer system is compatible with Taq DNA polymerase and reverse transcriptase, enabling seamless reverse transcription and amplification in a single reaction.Key Features:✔ Visual tracking: Yellow dye (non-inhibitory) confirms reagent addition, preventing omissions✔ Contamination control: dUTP/UDG system eliminates carryover amplicon contamination✔ Broad applications: Suitable for one-step RT-qPCR (RNA targets) and hot-start DNA amplification✔ Optimized performance: High sensitivity and reproducibility for low-abundance targetsProtocolI. Reaction SetupReagent PreparationThaw all components at room temperature or 4°C.Mix gently and centrifuge briefly before use.Recommendations:Protect probes from light.Aliquot master mix to avoid freeze-thaw cycles.Prepare Reaction MixComponentVolume per 25 µLVolume per 50 µLFinal ConcentrationStableMax RT-PCR Master Mix (Dye, UDG, One Tube)10 µL20 µL1×Primer-probe Mix1 µL2µL/Templates5 µL10 µL/ddH2OUp to 25 µLUp to 50 µL/Total25 µL50 µL/*Note:Primer/template volumes can be adjusted.For multiplex assays, optimize primer concentrations empirically.Mixing:Gently pipette mix or vortex briefly.Centrifuge at room temperature to collect liquid.PCR Amplification:Load tubes onto a thermal cycler with heated lid (105°C recommended).II. Thermal Cycling ProgramStepTemperatureTimeCycles150℃15min1295℃2min 30s1394℃10s45455℃40s（Fluorescence read）45Optimization Guidelines:a. Reverse Transcription: 5–15 min (adjust based on target abundance).b. Cycle Number: Reduce to 40 cycles for high-copy targets.c. Primer Design:Final concentration: 0.2 µM (typical).Tm ≥55°C (use software for accurate calculation).Adjust annealing temperature (±3°C) if amplification is suboptimal