Extraction-Free RT-PCR Master Mix, Strong Formulation, TaqMan Probe (#149)

Description

The Master Mix is used for real-time qualitative and quantitative RT-PCR amplifications with TaqMan Probe dye for extraction-free RNA samples. The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers and RNA template.

This product, RT-PCR 149, contains a stronger formulation of 50% more enzymes than RT-PCR 146.

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 master mix contains extra more redundant amounts of the RTase and DNA polymerase, specifically designed to overcome endogenous inhibition in extraction-free RNA samples.

• 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 a complementary DNA strand on RNA template from a gene-specific primer, ≤1 unit per 20μL of reaction.

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

• The kit also contains Taq-Fast DNA polymerase which extends more than 300 bases with short cycling program.

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

• The preferable PCR product size is ≤150bp.

• The kit has three formulations of No ROX, Low ROX or High ROX concentrations 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. 1.Harvest 10,000–100,000 cells.

2. 2.Add lysis buffer and incubate for 5 minutes.

3. 3.Heat at 95°C for 10 minutes.

4. 4.Add lysate directly to RT-PCR setup.

5. 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.

Product Manual

User Manual