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 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
