PanGreen™ One-Step RT-qPCR Kit

PanGreen™ One-Step RT-qPCR Kit
PanGreen™ One-Step RT-qPCR Kit
PanGreen™ One-Step RT-qPCR Kit

Catalog numberQSR01-0100

Size100 reactions (20 μl vol)


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.

Kit Contents

2X Universal SYBR® Green Master Mix1 ml x 1 vial
RScript Enzyme Mix20 μl x 1 vial
  • Enhanced RScript reverse transcriptase with RNase inhibitor.
  • Compatible with nano taq DNA polymerase to form one tube RT-qPCR.
  • Maximizes product yield with nano technology.
  • Compatible with the majority of qPCR systems.
Reaction Setup

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 ReactionVolume per 10 μl ReactionFinal Concentration
2X Universal SYBR® Green Master Mix10 μl5 μl1x
RScript Enzyme mix (RScript reverse transcriptase & RNase inhibitor)0.2 μl0.1 μl1x
Forward and reverse primersVariableVariable300 nM* each
RNA (add at step 4) VariableVariableTotal RNA: 1 ng-5 μg
Nuclease-free H2OVariableVariable
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.

  • Process in the thermal cycler for 35-45 cycles in Table 2. View More↘

Table 2. Thermal Cycling Protocol

cDNA Synthesis42°C15 minutes1
Pre-Denaturation95°C5 minutes1
Denaturation95°C10 seconds35-45
Annealing60°C60 seconds
Instrument Cooling40°C10 seconds1

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:

  • Shake gently before use to avoid foaming and low-speed centrifugation.
  • During operation, always wear a lab coat, disposable gloves, and protective equipment.
Required Materials but Not Provided
  • A compatible real-time PCR instrument
  • Vortex or equivalent
  • Microcentrifuge
  • Plates and seals for your instruments

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.

Performance of PanProbes™ One-Step RT-qPCR Master Mix.Figure 1. Performance of PanProbes™ One-Step RT-qPCR Master Mix.

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