TOROIVD TECHNOLOGY
COMPANY LIMITED
Evolving Enzymes
Inovating IVD
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.
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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.
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TOROGreen® HRM qPCR Master Mix 10μl |
Premix and Loading |
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Template DNA Dilutions 2μl |
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2μM Forward primer 4μl |
Premix and Loading |
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2μM Reverse primer 4μl |
-Gently mix the reaction solutions and spin down in microcentrifuge.
Cycling conditions
For qPCR applications
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2-Step Cycle |
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Pre-denaturation ： 95°C 60sec 1cycle |
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Denaturation ： 95°C 10sec 40cycles |
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Extension /Annealing ： 60°C 30sec 40cycles |
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Data collection should be performed at the extension step. |
For HRM analysis
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3-Step Cycle |
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Pre-denaturation ： 95°C 60sec 1cycle |
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Denaturation ： 95°C 15sec 40cycles |
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Annealing ： 55°C-65°C 15sec 40cycles |
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Extension ： 72°C 45sec 40cycles |
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Data collection should be performed at the extension step. |
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HRM analysis Cycle |
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95°C 60sec 1cycle |
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40°C 60sec 1cycle |
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65°C 1sec 1cycle |
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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
