PanProbes™ One-Step RT-qPCR Kit

Catalog numberQPR01-0100

Size100 reactions (20 μl vol)

Concentration2X

StorageStable for up to 3 months at 4°C. Stable for up to 24 months at -20°C.

$132Inquire

Description

PanProbes™ 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 qPCR Master Mix is a 2x concentrated, ready-to-use master mix optimized for probe-based real-time PCR and compatible with the majority of commercially available real-time PCR systems (ROX-independent and ROX-dependent). It contains antibody-mediated hot-start Taq DNA polymerase, dNTPs, MgCl₂, enhancers, stabilizers and essentials for a success PCR reaction.

Kit Contents

2X Universal qPCR Master Mix	1 ml x 1 vial
RScript Enzyme Mix	20 μl x 1 vial

Features

Enhanced rscript reverse transcriptase with RNase inhibitor.
Optimized for probe-based real-time PCR.
Compatible with the majority of qPCR systems.

Reaction Setup

1. Thaw RScript Enzyme mix, 2X Universal qPCR 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 qPCR 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
Fluorogenic probe(s)	Variable	Variable	150–250 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), 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.

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

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	35-45
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:

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

Performance

To evaluate the performance, the Ribonuclease P (RP)gene with varying RNA template concentrations, through serial dilutions, were used. Based on the fluorescence value of the RP probe, the results show the performance corresponding to the Ct value with different copy numbers.

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

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