2×Probe qPCR Mix

Product Composition

* Used to correct fluorescence signal errors between wells.

 Applied Biosystems 5700,7000,7300,7700,7900,7900HT,7900HT Fast,Applied Biosystems StepOne™,StepOnePlus™ When other fluorescence quantitative PCR instruments require the addition of high-concentration ROX Reference Dye, the amount of 50×ROX Reference Dye added is 1/50 of the PCR reaction system; Applied Biosystems 7500, 7500 Fast, ViiA™7, Stratagene MX4000™, MX3005P™, MX3000P™ and others require the addition of low concentration ROX Reference Dye When using a fluorescence quantitative PCR instrument, the amount of 50 × ROX Reference Dye added 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. Available for PCR:Bio-Rad CFX96TM,CFX384TM,iCycler iQTM,iQTM5,MyiQTM,MiniOpticonTM,Opticon ®,Opticon 2,Chromo4TM,Cepheid SmartCycler®,Eppendorf Mastercycler®ep realplex ,realplex 2, Illumina Eco qPCR ,Qiagen/Corbett Rotor-Gene®Q,Rotor-Gene® 3000,Rotor-Gene® 6000,Roche Applied Science LightCyclerTM 480,Thermo Scientific PikoReal Cycler and so on

Product storage and validity period 

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

Product Introduction

2×Probe qPCR Mix is a twice-concentration premix specially designed for probe-based real-time fluorescence quantitative PCR. The product uses anti-Taq antibody hot-start DNA polymerase, combined with the optimal Buffer for Real Time PCR, which can greatly improve the amplification efficiency of PCR and perform highly sensitive Real Time PCR amplification reactions. The product is separately configured with ROX dye and is suitable for use with mainstream  fluorescence quantitative PCR instruments in the market such as Applied Biosystems, Bio-Rad, Eppendorf, Roche, etc. This product is suitable for rapid Real

Time PCR amplification reaction. It can obtain a good standard curve in a wide quantitative area, accurately quantify and detect target genes, and has good repeatability and high reliability.

Reagents and items that users need to prepare

1. Primers and probes, DNA or cDNA template.

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

 3. Micropipette and clean tips.

4. Real Time PCR instrument (authorized instrument).

Notes

1. The2×Probe qPCR Mix stored at -20 can be  dissolved slowly by hand before use, and gently mixed up and down until all precipitation disappears.

2. When preparing the PCR reaction solution, please place the reagents on ice and avoid exposure to strong light.

 3. Avoid repeated freezing and thawing Repeated freezing and thawing may cause product performance to decrease.

4. When preparing the reaction solution, please use clean pipette tips (suction tips with filter elements recommended) and centrifuge tubes to prevent contamination as much as possible.

• Applied Bio systems 7300/7500/7500 Fast Real-Time PCR System and Step One PlusTM

How to operate the Real-Time PCR System

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

aa

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

*2 When using the 7500 Real-Time PCR System and 7500 Fast Real-Time PCR System, the amount of 50×ROX Reference Dye added is the amount of the PCR reaction system.

1/250. When using ABI PRISM 7300 Real-Time PCR System and Step One PlusTM, the added amount of 50×ROX Reference Dye is the amount of the PCR reaction system.1/50

*3 In a 20 ul reaction system, the amount of DNA template added is usually less than 100 ng. Because different types of DNA templates contain different copy numbers of target genes, it can be

Carry out gradient dilution to determine the optimal amount of DNA template to add. If you want to use this product to perform the second PCR amplification reaction of the two-step RT-PCR reaction, the first step

When RT reaction solution is used as DNA template, the amount added should not exceed 10% of the total volume of the PCR reaction solution.

*4 Prepare the reaction solution according to the recommended system for each instrument.

2.       Perform Real Time PCR reaction

It is recommended to use the following two-step PCR reaction procedure. If this procedure does not produce good experimental results, then optimize the PCR conditions.

When the amplification performance of the two-step PCR reaction is poor due to the use of primers with low Tm values, etc., you can try the three-step PCR amplification reaction.

1)      <Applied Biosystems 7300/7500 and Step One PlusTM Real-Time PCR System> Two-step PCR amplification standard procedure:

Stage 1: Pre-denaturation

Repsÿ1

95℃ 30 seconds

Stage 2: PCR reaction

Repsÿ40

95℃  5 seconds

60℃  30ÿ34 seconds*

* When using Step One PlusTM, please set it to 30 seconds; when using 7300, please set it to 31 seconds; when using 7500, please set it to 34 seconds.

2)      <7500 Fast Real-Time PCR System> two-step PCR amplification standard procedure:

Stage 1: Predenaturation

Repsÿ1

95℃ 30 seconds

Stage 2: PCR reaction

Repsÿ40

95℃  3 seconds

60℃  30 seconds

3.      Analysis of experimental results

After the reaction, confirm the amplification curve of Real Time PCR and prepare a standard curve when performing quantitative PCR. For analysis methods, please refer to the operation of the instrument.Work manual.

• How to operate the LightCycler®/LightCycler®480 System Real Time PCR instrument

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

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

*2 In a 20 ÿl reaction system, the amount of DNA template added is usually less than 100 ng. Because different types of DNA templates contain different copy numbers of target genes, it can be

Carry out gradient dilution to determine the optimal amount of DNA template to add. If you want to use this product to perform the second PCR amplification reaction of the two-step RT-PCR reaction, the first step

When RT reaction solution is used as DNA template, the amount added should not exceed 10% of the total volume of the PCR reaction solution.

2.     Perform Real Time PCR reaction

For PCR reaction capillaries, please centrifuge them at low speed for a few seconds and put them into the LightCycler for Real Time PCR reaction. It is recommended to adopt the following

If the two-step PCR reaction program listed below does not produce good experimental results, then optimize the PCR conditions. Since using Tm value

When the amplification performance of the two-step PCR reaction is poor due to lower primers and other reasons, you can try a three-step PCR amplification reaction.

1)      <LightCycler®> two-step PCR amplification standard procedure:

Stage 1: Pre-denaturation

1 Cycle

95℃  30 seconds 20℃ /second

Stage 2: PCR reaction

40℃  Cycles

95℃  5 seconds 20℃ /second

60℃  20 seconds 20℃ /second

2)      <LightCycler®480 System> Two-step PCR amplification standard procedure:

Denaturation: (1 cycle)

95℃ 30 seconds (heating rate 4.4℃ /second)

PCR: Analysis mode: Quantitative analysis (40 cycles)

95℃  5 seconds (heating rate 4.4℃ /second)

60℃  30 seconds (heating rate 2.2℃ /second, Acquisition Mode: Single)

3)      Analysis of experimental results

After the reaction, confirm the amplification curve of Real Time PCR and prepare a standard curve when performing quantitative PCR. For analysis methods, please refer to the operation of the instrument.

Work manual.