【Description】

• Probe qPCR Master Mix is a Taq DNA polymerase-based 2× master mix for realtime PCR, which contains all components, except for the primer and probe. This reagent is applicable in TaqMan assays or hybridization probe assays, in combination with each probe. This reagent can be used in glass capillary systems or passive reference system. Hot Start technology with anti-Taq DNA polymerase antibodies enables high specificity and reproducible amplification. The specially optimized PCR buffer make the mix more efficient amplification of GC-rich templates and more stable at room temperature.

【Feature】

• -High specificity: High efficiency Taq antibodies and optimized PCR buffer greatly reduce the formation of primer dimers, so it easily meets MIQE requirements.

• -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】

•  QPT-100 can be used for 500 reactions for a total 20µL reaction volume.

• Cat NO.	Components	Size
  QPT-100	Probe qPCR Master Mix	1 mL ×5tubes/ bag

【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/Probe Design】

• -Design of primers

  Primer length: 18–25bp；Tm of primer: 60–65°C；GC content: 40–80%；Target length: 70–200 bp; 

  Larger targets (>200 bp) tend to reduce the efficiency and specificity of amplification.

  Purification grade: OPC or HPLC grade ；

• -Design of probes

  Probe length: 20–30bp；Tm of probe: 65–70°C；GC content: 40–60%；

  Purification grade: HPLC.

• -Checking the performance of primers and probes：

  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 probe, and draw a standard curve. Confirm that the PCR efficiency is between 90% and 110% and R2 is equal to or greater than 0.99. If the PCR efficiency or R2 are outside of these ranges, the reaction conditions should be optimized. If this does not improve the result, the primers and/or probes 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】

【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:   Probe qPCR Master Mix  (QPT-100)-English-

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

•     MSDS： Material Safety Data Sheet for QPT-100

•     COA： Certificate of Analysis for QPT-100   

    

【Order information】