PanProbes™ Universal qPCR MasterMix

Catalog numberQPD01-0100

Size100 reactions (20 μl vol)

Concentration2X

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

$48Inquire

Description

PanProbesTM 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

1ml x 1 vial

Features

Enhanced antibody-mediated hot-start DNA polymerase.
Optimized for probe-based real-time PCR.
Compatible with the majority of qPCR systems.

Reaction Setup

1. Thaw PanProbes™ 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 DNA 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
PanProbes™ Universal qPCR Master Mix (2x)	10 μl	5 μl	1x
Forward and reverse primers	Variable	Variable	300-500 nM each primer
Fluorogenic probe(s)	Variable	Variable	150–250 nM each
DNA template(add at step 4)	Variable	Variable	cDNA: 1 pg-10 ng Genomic DNA: 50 ng-250 ng
Nuclease-free H2O	Variable	Variable	—
Total reaction mix volume	20 μl	10 μl	—

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 DNA samples (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

Initial Denaturation	3-5 minutes at 95°C (5 mins for GC rich or complex templates)
Denaturation	15 seconds at 95°C
Annealing & Extension	60 seconds at 60°C and Plate Read

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

The SARS-CoV-2 nucleocapsid (N1) gene with serial diluted DNA template concentrations are prepared and conducted by qPCR. Based on the detected N1 probe 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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