Catalog numberQSR01-0100
Size100 reactions (20 μl vol)
Concentration2X
StorageStable for up to 3 months at 4°C. Stable for up to 24 months at -20°C.
Catalog numberQSR01-0100
Size100 reactions (20 μl vol)
Concentration2X
StorageStable for up to 3 months at 4°C. Stable for up to 24 months at -20°C.
PanGreen™ One-Step RT-qPCR Kit delivers high sensitivity of the target RNA level due to its RScript reverse transcriptase, a reduced RNase H+ activity MMLV enzyme in addition to a powerful RNase inhibitors mix which aim to diminish RNA degradation and mispriming during reaction setup and reverse transcription to guarantee optimal RT efficiency.
The Universal SYBR® Green Master Mix is a 2x concentrated, ready for use Master Mix reaction enhanced for dye-based quantitative PCR (qPCR) and compatible with the majority of commercially available real-time PCR systems (ROX-independent and ROX-dependent). It contains hot-start Taq DNA polymerase, dNTPs, MgCl2, SYBR® Green I dye, enhancers, stabilizers and essentials for a success PCR reaction.
2X Universal SYBR® Green Master Mix | 1 ml x 1 vial |
RScript Enzyme Mix | 20 μl x 1 vial |
1. Thaw RScript Enzyme mix, 2X Universal SYBR® Green Master Mix and the rest of frozen reaction components to a temperature of 4°C. In order to entirely collect solutions, combine thoroughly and centrifuge briefly, then store at 4°C and avoid from light.
2. Prepare (on ice or at room temperature) enough assay Master Mix for all reactions by adding all necessary components, except the RNA template, according to the recommendations in Table 1. View More↘
Table 1. Reaction Setup
Component | Volume per 20 μl Reaction | Volume per 10 μl Reaction | Final Concentration |
---|---|---|---|
2X Universal SYBR® Green Master Mix | 10 μl | 5 μl | 1x |
RScript Enzyme mix (RScript reverse transcriptase & RNase inhibitor) | 0.2 μl | 0.1 μl | 1x |
Forward and reverse primers | Variable | Variable | 300 nM* each |
RNA (add at step 4) | Variable | Variable | Total RNA: 1 ng-5 μg |
Nuclease-free H2O | Variable | Variable | — |
Total reaction setup volume | 20 μl | 10 μl | — |
Note: Optimization may be needed for better performance.
3. Combine the assay Master Mix thoroughly to ensure consistency and equally dispense the solution into each qPCR tube or into the wells of a qPCR plate. Employ good pipetting practice to ensure assay precision and accuracy.
4. Add RNA template (and DNase-free H2O if needed) to the PCR tubes or wells containing assay Master Mix (Table 1), seal the tubes or wells with flat caps or optically transparent film. Note: to ensure thorough mixing of reaction components, vortex for approximately 30 seconds (or more).
5. Spin the tubes or plate to remove any air bubbles and collect the reaction mixture in the vessel bottom.
6. Setup the thermal cycling protocol on a real-time PCR instrument according to Table 2.
(Note: optimization may be needed for better performance).
7. Load the PCR tubes or plate into the real-time PCR instrument and commence the run.
8. Perform data analysis according to the instrument-specific instructions.
Table 2. Thermal Cycling Protocol
Steps | Temperature | Time | Cycle(s) |
cDNA Synthesis | 42°C | 15 minutes | 1 |
Pre-Denaturation | 95°C | 5 minutes | 1 |
Denaturation | 95°C | 10 seconds | 35-45 |
Annealing | 60°C | 60 seconds | |
Instrument Cooling | 40°C | 10 seconds | 1 |
Note: Optimal conditions for amplification will vary depending on the primers and thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler
Important notes:
The VP1 capsid gene of EV71 virus with serial diluted RNA template concentrations are prepared and conducted by qPCR. Based on the detected SYBR Green fluorescence values, the result shows the Ct values corresponding to the different concentration.
Figure 1. Performance of PanProbes™ One-Step RT-qPCR Master Mix.
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