【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 Hot Start Taq DNA Polymerase, PCR Buffer, dNTPs, Super EvaGreen®dye, ROX, Enhancer and Stabilizer. Except for using Super EvaGreen® instead of SYBR Green I, the master mix has the same composition as TOROGreen® qPCR Master Mix (QST-100). Therefore, it has all the features of QST-100. Due to the use of Super EvaGreen®, HRM analysis can be performed. Moreover, all primers will not form dimers in the NTC tests.

【Feature】

• -No primer dimer: all primers will not form dimers in the NTC tests.

• -HRM analysis: HRM analysis can be performed with the master mix.

• -Room-temperature stable: the performance is not easily decrease during storing and shipping. 

• -Wide dynamic range: the master mix demonstrates excellent reproducibility over a wide dynamic range and provides efficient amplification over 8 logs of sample input.

【Components】

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

• Cat NO.	Components	Size
  QET-100	TOROGreen® HRM qPCR Master Mix	1 mL ×10tubes/ Kit

  
  

  
  

【Storage】

• This reagent can be stored at 2-8°C for 12 months and protected from light. 

• For longer storage, this reagent should be kept at -20°C and protected from light. 

【Primer Design】

• - Primer length: 18~30bp - GC content of primer: 40~80%

• -Target length: ≤ 200 bp (optimally, ≤ 150bp) 

 -Checking the primers:

• -Prepare a dilution series with five or more dilutions of template DNA. Perform qPCR assay using the diluted DNA with the newly designed primers and draw a standard curve. Confirm that the PCR efficiency is between 95% and 105% and R2 is equal to or greater than 0.99. If the PCR efficiency or R2 are outside of these ranges, the primers concentration and reaction conditions should be optimized. If this does not improve the result, the primers should be redesigned.

【Template DNA】

• -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. 

• -cDNA: Reverse transcription reactions from total or poly (A)+ RNA may be used directly, or after dilution for realtime PCR. Before the reverse transcription reaction, it is essential to assess the extent of genomic DNA contamination with no-reverse transcription control. If genomic DNA contamination affects the Cq values, it is essential to be eliminated by DNase treatment.

【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 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).

【Example】

• The genomic DNA of the Aope gene model mice was amplified using QET-100. The results of the melting curve were used to genotype the mice, as shown in Figures 1-a and 1-b:

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• Result: From the melting curve, it shows that the fluorescence values are divided into three peaks, representing wild-type, heterozygous, and homozygous mice, based on the differences in DNA template binding with Super EvaGreen dye.

• Conclusion: By adding the saturated fluorescent dye Super EvaGreen, QET-100 can perform high-resolution melting curve analysis, replacing the use of partial probe fluorescence quantification methods such as SNP sites recognition and gene typing, greatly reducing experimental costs.

【References】

•  Bustin SA, Benes V, Garson JA, etc,al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. ClinChem.2009,55(4):611-22.

【Documents】

•     Manuals:   TOROGreen® HRM qPCR Master Mix (QET-100)-English-

•     Brochures:   Real-time PCR Premix for gene expression analysis

•     MSDS： Material Safety Data Sheet for QET-100

•     COA： Certificate of Analysis for QET-100   

    

【Order information】