Hifair™ Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR kit based on RNA as template. In the process of the experiment, reverse transcription and quantitative PCR were carried out in the same tube, which simplified the experimental operation and reduced the risk of contamination.

In this kit, the first strand cDNA was efficiently synthesized by heat-resistant Hifair^TM V Reverse Transcriptase and quantitatively amplified by UNICON™ HotStart Taq DNA Polymerase. The kit mainly contains optimized MP buffer, enzymes mix, etc. The buffer solution already contains Mg²⁺ and dNTP. In addition, the factors that can effectively inhibit the non-specific PCR amplification and improve the amplification efficiency of multiple qPCR reactions are added, which can ensure the amplification efficiency and carry out up to multiple amplification reaction. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.

Features

1. Good versatility: It is suitable for qPCR amplification and detection of nucleic acids of pathogenic microorganisms, human genomes, plants and other species.

2. High sensitivity: The carefully optimized buffer system improves the detection sensitivity of low-concentration templates up to 250 copies/mL.

3. dUP/UDG system: dUTP/ Heat-labile UDG anti-pollution system is introduced to degrade aerosol pollution of products efficiently, reduce false positive, and ensure authentic results.

4. Supporting fast program: compatible with fast 40 min program.

5. Super stability: stable at 37℃ for 14 days,repeated freezing and thawing 10 times.

Components

Note: 1) 5×Hifair^TM MP Buffer is the abbreviation of Hifair^TM V Multiplex One Step RT-qPCR Probe Buffer, which includes dNTPs, dUTP, Mg²⁺, stabilizers, enhancers, and more.

2) Hifair Enzyme Mix mainly contains heat-resistant Hifair^TM V reverse transcriptase, UNICON^TM HotStart Taq DNA polymeras and UDGase.

3) GC enhancer is optional. Please find the GC enhancer free version: Cat: 16901ES.

4) The 16630-A and 16630-B is equivent to 16901-A and  16901-B, respectivrly.

Specifications

Storage

The product should be stored at -25°C~-15°C for 1 year.

Figures

Figure 1. High detection Rate

Instructions

1. Reaction Composition

Note：Be sure to mix well before use, avoid excessive bubbles caused by violent vibration.

a) Primer concentration: Primer mix including multiplex primer, depending on the situation optimal primer concentration may be between 0.1 and 1.0 μM.

b) Probe concentration: Probe mix including multiplex probe labeling difference fluorescent group, depending on the situation optimal probe concentration may be between 0.05 and 0.5 μM.

c) Template dilution: qPCR is highly sensitive and it is recommended to dilute the template. The control Ct value is suitable between 20 and 35.

d) System preparation: un head with filter element. Avoid cross contamination and aerosol contamination.

2. Optimized Cycling Protocol

Note：a) Reverse transcription: The temperature can select 42°C or 50°C for 10-15 minutes.

b) Amplification reaction: The temperature is adjusted according to the Tm value of the designed primers.

c) Fluorescence signal acquisition: Please set the experimental procedure according to the requirements of the instrument manual.