2×One Step Probe RT-qPCR Mix

Product Composition

*1 Including dNTP Mix, Mg 2+, etc.

*2 Contains reverse transcriptase, Taq enzyme, Taq enzyme antibody, RNase Inhibitor, etc.

*3 Used to correct for fluorescence signal errors between wells.

Storage and Validity Period

The validity period is more than 2 years when stored at- 20°C

Introduction

This product is a special reagent for Real Time One Step RT-PCR using the probe method. Using this product for Real Time RT- PCR reaction can continuously perform reverse transcription and qPCR amplification in the same reaction tube. It is simple to operate and can effectively prevent contamination. This reaction system can monitor the amplification product in real time, greatly improves the detection sensitivity, and omits the electrophoresis step after the PCR reaction, making it very suitable for the detection of RNA viruses.

This product uses high-efficiency reverse transcriptase and highly specific hot-start Taq DNA polymerase to perform stable and efficient Real Time One Step RT-PCR reactions. For real time fluorescence quantitative PCR instruments that use ROX as the calibration dye, this product is also equipped with ROX dye to correct the fluorescence signal errors generated between the wells of the quantitative PCR instrument.

Reagents and items that users need to prepare by themselves

1.  PCR primers and probes.

2.  RNA template.

3.  Suitable for single tube, 8-strip tube, or 96-well PCR tube (plate) for real time         fluorescence  quantitative PCR.

 4. Micropipettes and clean tips with filters.

5. Real Time PCR instrument (authorized instrument).

Notes

1. When several Real Time One Step Probe RT-PCR reactions need to be performed at the same time, a mixture of various reagents (Master Mix; including RNase-free Water, Buffer, enzyme, etc.) should be prepared first, and then divided into each reaction tube. This can make the reagent volume taken more accurate, reduce reagent loss, and avoid repeated dispensing of the same reagent. At the same time, it can also reduce errors caused by experimental operations or experimental samples.

2. When using RT-Taq enzyme mixture, mix gently to avoid foaming; carefully centrifuge and collect to the bottom of the reaction tube before dispensing; because the enzyme storage solution contains high concentration of glycerol and high viscosity, dispensing It should be absorbed slowly.

3. When preparing and dispensing the reaction solution, be sure to use new filter tips, centrifuge tubes, etc. to avoid contamination as much as possible.

 4. This product can only use specific reverse transcription primers. Random Primer and Oligo dT Primer cannot be used. Reverse transcription reaction.

Steps

1.     Prepare RT-qPCR reaction solution according to the following components (please prepare the reaction solution on ice)

*1 the final concentration of primers and probes is 0.2 µM to obtain better results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1 to 1.0 µM.

*2 Used to correct for fluorescence signal errors between wells. Use Applied Biosystems 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast;When using Applied Biosystems StepOne™, StepOnePlus™ and other fluorescence quantitative PCR instruments that require the addition of high-concentration ROX Reference Dye, 50 × ROX

The amount of Reference Dye added is 1/50 of the PCR reaction system; Applied Biosystems 7500, 7500 Fast, ViiA™7, Stratagene MX4000™,

When using MX3005P™, MX3000P™ and other fluorescence quantitative PCR instruments that require the addition of low concentration ROX Reference Dye, the addition of 50 × ROX Reference DyeThe amount is 1/250 of the PCR reaction system;

The following are fluorescence quantitative PCR instruments that do not require the addition of ROX Reference Dye: Bio-Rad CFX96™, CFX384™, iCycler

iQ™ IQ™5 MyiQ™ ,MiniOpticon™ ,Opticon®, Opticon 2 Chromo4™, Cepheid SmartCycler®, Eppendorf Mastercycler®ep  realplex,realplex 2,Illumina Eco qPCRÿQiagen/Corbett Rotor-Gene®QÿRoto,r-Gene® 3000ÿRotor-Gene® 6000,Roche AppliedScience LightCyclerTM 480,Thermo Scientific PikoReal Cycler

*3 Recommended to use 20 pg~200 ng Total RNA as template in 50 µl reaction solution.

*4 Determine the volume of the reaction system according to the requirements of different instruments.

2.  Perform Real Time One Step RT-PCR reaction

Please use a centrifuge to centrifuge the PCR reaction tube briefly and then put it into a fluorescence quantitative PCR instrument for Real Time PCR reaction. Recommended to use the following diagram

The table shows the standard PCR reaction procedures. If good experimental results cannot be obtained using this procedure, optimize the PCR conditions.

3. Analysis of experimental results

After the reaction, confirm the amplification curve of Real Time One Step RT-PCR, and prepare a standard curve when performing RT-PCR quantification.

For analysis methods, refer to the instrument's operating manual.