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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 enables high specificity and reproducible amplification.


COMPONENTS

 

QET-100 can be used for 1000 reactions for a total 20µL reaction volume.

 

Cat NO.

Components

Size

QET-100

2×TOROGreen®HRM qPCR Master Mix  

1 ml ×10tubes/ Kit

 

PRIMER DESIGN

 

- Primer length: 20~30 mer
GC content of primer: 40~80%
-
Target length: ≤ 200 bp (optimally, ≤ 150bp) 

 

SPECIMEN

 

-cDNA :  Reverse transcription reactions from total or poly (A)+ RNA may be used directly, or after dilution, for realtime PCR. Purified RNA 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. 

 

DETECTION


-This reagent with 1× 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); Mx3000P,3005P,MX4000,etc.(Agilent).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);


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.

 

Components                20μL reaction ×n 

Operation

TOROGreen®HRM qPCR Master Mix              10μL×n

Premix and Loading

8μM Reverse primer            1μL×n

Premix 

8μM Reverse primer            1μL×n

Template DNA Dilutions                                    8μL×n

Premix and Loading

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


CYCLING CONDITIONS

 

For qPCR applications(2-Step Cycle)

1

Pre-denaturation

95℃

60 sec

1cycle

2

Denaturation

95°C

10 sec

40

cycles  

Annealing/ Extension

60°C

30 sec

Data collection should be performed at the extension step.  

 

 For HRM analysis  (3-Step Cycle)

1

Pre-denaturation

95℃

60 sec

1cycle

2

Denaturation

95°C

15 sec

40

cycles

Annealing

55-65°C

15 sec

 Extension

72°C

45 sec

 

 

3

HRM analysis Cycle

 95°C 

   60sec  

1cycle

  40°C  

   60sec  

1cycle

   65°C  

1sec 

1cycle

 97°C 

    1sec    

1cycle

Data collection should be performed at the extension step.  

 

 

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 are employed, the recommended conditions from each company should  be used. 

-HRM analysis Cycle conditions are for reference only and shall be recommended according to the instructions of the instrument manufacturer.

 

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 





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