PanGreen™ One-Step RT-qPCR Kit

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

• 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.

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↘

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↘

• 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.

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