Cat. No. / ID: P7730L
The StableScript™ One-Step RT-qPCR Mix is a highly efficient and sensitive RT-qPCR formulation for DNA and RNA detection using
hydrolysis probes. The 10X enzyme mix contains a blend of a new RNAse H minus reverse transcriptase with higher
thermostability and inhibitor resistance (StableScript™), a fast-activating hot start DNA polymerase (Phoenix™), and specific
enhancers to increase sensitivity and inhibitor resistance.
StableScript™ One-Step RT-qPCR Mix is supplied with 4X StableScript™ Reaction Buffer B7720L, containing dNTPs and
Magnesium.
StableScript™ One-Step RT-qPCR Mix is a blend of StableScript™ Reverse Transcriptase and Phoenix™ Hot Start Taq DNA
Polymerase enzymes. StableScript™ Reverse Transcriptase is engineered for increased thermostability. Increasing the temperature
for cDNA synthesis can aid in overcoming challenging RNA targets because higher temperatures help unravel RNA structures,
reducing the likelihood of hindering reverse transcription.
The Phoenix™ Polymerase is a recombinant, thermostable Taq DNA polymerase complexed with a thermolabile, neutralizing
antibody. This fast-activating antibody-mediated hot-start capability enhances the overall specificity, sensitivity, and yield of the PCR
reaction by reducing nonspecific amplification and primer-dimer formation prior to cycling. Together with StableScript™, Phoenix™
creates StableScript™ One-Step RT-qPCR Mix, an extremely sensitive RT-qPCR mix:
Mix properties
StableScript™ One Step RT-qPCR reaction setup
General precautions against degradation of RNA template should be taken when setting up a reaction, including setting up the
reaction with nuclease free water, RNase inhibitor, nuclease free PCR tubes and sterile pipette tips with filter, adding reverse
transcriptase last, gentle pipetting, thorough mixing and a quick centrifugation. The following procedure can be used as a
guideline. Reactions may need to be optimized individually.
Components | Volume/Rxn | Final Concentration |
---|---|---|
4X StableScript™ Reaction Buffer | 5μL | 1X |
Primer/Probe Mix | XμL | Variable |
10X StableScript™ Enzyme Mix | 2μL | 1X |
RNA Template | Up to 2μL | 1pg to 1µg total RNA |
Nuclease-Free Water | To a final 20μL reaction volume | - |
Thermal Cycling Conditions:
Program the cycling conditions are recommended as below.
Standard Cycling Program* Steps | Temperature | Time | Cycles |
Reverse Transcription | 55°C | 10 min | 1 |
Taq Activation/Initial Denaturation | 95°C | 3 min | 1 |
Denaturation | 95°C | 5-10 sec | 40 |
Annealing/Extension* | 60°C | 30-60 sec |
*Cycling parameters can be modified (especially the annealing/extension condition) to fit specific assays.
Quality control analysis
The functionality of the RT-PCR Assay is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The
amplification threshold (Ct) of the test lot is compared to a reference lot.
Notes:
Enzyme components were tested prior to formulation of the master mix and found free of contaminating endonucleases and
exonucleases. Enzyme purity was >99% as determined by SDS-PAGE, and negligible E. coli genomic DNA contamination was
confirmed by qPCR. Specific activity was verified for each enzyme pre-formulation.
This product is available for molecular biology applications such as: