TOROIVD TECHNOLOGY 

    COMPANY LIMITED

Evolving Enzymes  

   Inovating IVD

MORE
PRODUCT CENTER
TOROGreen® HRM qPCR Master Mix
    Publish time 2020-08-18 17:45    

TOROGreen® HRM qPCR Master Mix is a Taq DNA polymerase based 2 × master mix for use in qPCR applications and high-resolution melting (HRM) analysis, which contains the HotStart Taq DNA Polymerase, PCR Buffer, dNTPs, Super EvaGreen® dye, ROX, Enhancer and Stabilizer. Hot Start technology with anti -Taq DNA polymerase antibodies enableshigh specificity and reproducible amplification.


DESCRIPTION

TOROGreen® HRM qPCR Master Mix is a Taq DNA polymerase based 2 × master mix for use in qPCR applications and high-resolution melting (HRM) analysis, which contains the HotStart Taq DNA Polymerase, PCR Buffer, dNTPs, Super EvaGreen® dye, ROX, Enhancer and Stabilizer. Hot Start technology with anti -Taq DNA polymerase antibodies enableshigh specificity and reproducible amplification.

Components

The kit includes the following  reagents, and QET-100 can be used for 1000 reactions for a total 20ul reaction volume.

 

TOROGreen® HRM qPCR Master Mix                  1ml×10tube


Primer design

- Primer length: 20~30 mer
- GC content of primer: 40~60%
- Target length: ≤ 200 bp (optimally, ≤ 150bp) 


Specimen

-cDNA :  Reverse transcription reactions from total or poly (A)+RNA may be used directly, orafter dilution, for realtime PCR. Purified cDNA by phenol/ chloroform extraction and ethanol precipitation may also be used.  Oligo dT and random primers  are suitable for the reverse transcription reaction.
-Genomic DNA:  Purified DNA, which would be used for general PCR, is also suitable for real-time PCR. In the case of mammalian genomic DNA,  1~10 ng genomic DNA is sufficient for real-time PCR. 

Protocol 


-All solutions should be thawed at room temperature in dark, gently vortexed and briefly centrifuged.
-Prepare the following reaction in a thin-walled qPCR tube or plate at room temperature.

TOROGreen® HRM qPCR Master Mix        10μl

Premix and Loading

Template DNA Dilutions                          2μl

2μM Forward primer                               4μl

Premix and Loading

2μM Reverse primer                                4μl

-Gently mix the  reaction  solutions  and  spin down in microcentrifuge.


Cycling conditions
 

                                         For qPCR applications

2-Step Cycle

Pre-denaturation               95°C            60sec           1cycle

 Denaturation                    95°C            10sec           40cycles      

Extension /Annealing         60°C            30sec          40cycles

Data collection should be performed at the extension step. 


                                            For HRM analysis

3-Step Cycle

Pre-denaturation             95°C              60sec           1cycle

Denaturation                    95°C             15sec           40cycles      

Annealing                   55°C-65°C          15sec           40cycles 

Extension                        72°C             45sec           40cycles

Data collection should be performed at the extension step. 

HRM analysis Cycle

                95°C              60sec           1cycle

                          40°C              60sec           1cycle

                          65°C              1sec             1cycle

                                        97°C              1sec             1cycle

                                       Continuous collection(15Acq/ °C)

   

Notes 

-The annealing temperature can be set to 55~65°C, depending on the primer Tm value.
-The annealing time should be set for 5~20 seconds. Longer annealing time results inincreased efficiency, and a shorter  time decreases non-specific amplification.
-The pre-denaturation condition described above is sufficient  for inactivation of the anti-Taq DNA polymerase  antibodies   used in Hot Start PCR. To prevent unexpected  and inappropriate results, do not prolong the predenaturation period. 
-Data collection step should be longer than 10 sec.
-If commercially available primers or probes are employed, the recommended conditions from each company should  be used. 

Detection  

-This reagent can be used in general detection devices, not needing ROX such as: LineGene(bioer);LightCycler (Roche);  iCycler iQ,CFX96(Biorad/MJ); Thermal Cycler Dice(Takara); 
- This reagent with 1X ROX  can also be used in detection equipment using passive reference, such as:ABI PRISM® 7000, 7700, 7900 ,7300;Step One,Step one plus etc.(ABI) ,ABI PRISM® 7500, 7500Fast(ABI);
-This reagent  can not  be used in detection equipment using 0.1X ROX passive reference, such as:  Mx3000P,3005P,MX4000,etc.(Agilent) 

Storage

This reagent  his reagent can be stored at 4°C for 2 months and protected from light. For longer storage,  this reagent should be kept at -20°C and protected from light.      
 

Example 





HOW TO BUY PRODUCTS