Description

The 1-Step 2X Multiplex RT-PCR Master Mix is used for real-time qualitative and quantitative multiplex RT-PCR amplifications with TaqMan probes for up to four templates.

The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers, probes and RNA templates.

Kit Characteristics

Direct-from-sample: enables expression analysis directly from cultured cells without RNA purification.
Fast preparation: 5-minute lysis including DNase treatment versus 30–60 minutes for traditional RNA purification.

The kit is designed for multiplex RT-PCR with TaqMan probes.

For the reverse transcription step, this kit uses a highly efficient Thermophilic Reverse Transcriptase (US patent pending), which is a thermophilic type A polymerase, with optimal temperatures of 60-62°C.

The RTase is easily heat-inactivated at ≥90°C for 1min.

The RTase efficiently synthesizes complementary DNA strands on RNA templates from gene-specific primers at ≥1 unit per 20 μL of reaction.

The RTase reversely-transcribes single digit copies of target RNA molecules consistently.

The kit also contains Taq-Probe DNA polymerase, specially engineered for TaqMan probes and generating S-shaped curves.

Up to four pairs of gene-specific primers can be applied in one reaction.

The concentrations of the primers and probes are variable depending on assay designs and thermocycling protocols (User Manual Table 1).

The preferable PCR product size is ≤150bp.

The kit has three formulations of ROX, Low ROX or High ROX concentration for your choice.

Kit Components

2X Master Mix (2 × 1 mL / 5 × 1 mL / 10 mL for 200, 500, or 1000 × 20 μL reactions).
Lysis Buffer (2 × 10 mL / 1 × 50 mL / 1 × 100 mL).
User Manual.

Storage

Store at -20°C (stable up to 24 months with ≤10 freeze-thaw cycles). Transport at ≤4°C for up to 3 days.

Reaction Setup

20 μL Reaction: 10 μL Master Mix, primers, ≤4 μL RNA sample, water to 20 μL.
10 μL Reaction: 5 μL Master Mix, primers, ≤2 μL RNA sample, water to 10 μL.
Recommended amplicon size: ≤150 bp.

Compatible Instruments

Compatible with all major real-time PCR instruments, including but not limited to Applied Biosystems®, Bio-Rad®, Roche®, Qiagen®, Eppendorf®, and more.

Workflow

1.Harvest 10,000–100,000 cells.
2.Add lysis buffer and incubate for 5 minutes.
3.Heat at 95°C for 10 minutes.
4.Add lysate directly to RT-PCR setup.
5.Run RT-qPCR and analyze results.

Product Comparison

142: SYBR Green, No multiplexing, No probe system, Standard single-gene expression.

146: TaqMan, Yes multiplexing, Yes probe system, Multiplex TaqMan assays.

149: TaqMan, Yes (Super Multiplex), Yes probe system, Highly complex multi-target detection.

Q: What nucleotides are included in the 2× Probe qPCR Master Mix? Does it contain UTP?

A: The 2× Probe qPCR Master Mix contains all standard deoxynucleotides—dATP, dTTP, dGTP, and dCTP—each at a standard concentration of 200 µM.
UTP is not included by default, but custom formulations with UTP can be provided on request, depending on the required quantitative range.
Additionally, the mix uses a next-generation hot-start enzyme, which allows for higher reaction temperatures and results in enhanced dNTP stability and overall amplification performance.

Documents

User Manual

Extraction-free RT-PCR from cultured cells

Extraction-Free 1-Step 2X Multiplex RT-PCR Master Mix-TaqMan Probe (#146)-Biofargo