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| Product Name | Two-Component One-Step Multiplex RT-qPCR Mix for 25 µL RNA Reactions |
| Catalog No. | NATR-HMM-0220 |
| Description | Alta DiagnoTech one-step multiplex RT-qPCR mix completes reverse transcription and quantitative PCR in one reaction tube using separate buffer and enzyme components. |
| Intended Use | For singleplex or multiplex quantitative PCR using RNA templates. |
| Principle / Technology | Reverse transcription and real-time PCR occur sequentially in one tube. A UDG/dUTP system reduces the risk of carryover contamination from previous amplification products. |
| Sample Type | RNA template, 1 pg to 1 µg per 25 µL reaction |
| Reaction Conditions / Protocol | For a 25 µL reaction, combine 12 µL PCR buffer, 4 µL enzyme mix, 0.4 µL each of 10 µM forward and reverse primers, 0.2 µL of 10 µM hydrolysis probe, RNA template and RNase-free water to volume. Standard cycling: 55°C for 15 minutes, 95°C for 1 minute, then 45 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Fast cycling: 55°C for 5 minutes, 95°C for 1 minute, then 42 cycles of 95°C for 5 seconds and 60°C for 15 seconds. |
| Components / Formulation | PCR buffer and enzyme mix are supplied separately. The 100-, 1,000- and 5,000-reaction formats contain 1.2 mL, 12 mL and 5 × 12 mL buffer, plus 0.4 mL, 4 mL and 5 × 4 mL enzyme mix, respectively. |
| Storage Conditions | Store at -25°C to -15°C. |
| Package Specifications | Available in 100-, 1,000- and 5,000-reaction formats. |
| Shipping Conditions | Ship at 0°C or below. |
| Compatibility | Use a preliminary run to confirm that the real-time PCR instrument supports the fast cycling program. Adjust annealing/extension temperature for primer melting temperature and the target assay. |
| Application Notes / Precautions | Typical primer final concentration is 0.2 µM and may be optimized from 0.1-1.0 µM; each probe may be optimized from 50-300 nM. A Ct range of 20-35 is recommended after template dilution. Use RNase-free consumables, dissolve components fully, avoid vigorous mixing and bubbles, and mix and centrifuge reaction strips after template addition. Primer guidance: 18-30 nt, 40-60% GC, matched melting temperatures around 55-60°C and a 70-200 bp amplicon; avoid template secondary structure, primer self-complementarity, excessive 3'-end complementarity, more than three consecutive terminal G/C bases and a terminal T. Probe guidance: 18-30 nt, high-purity preparation, no 5'-terminal G, melting temperature around 65-70°C, mutations near the ends rather than the center, and minimal secondary structure. |
| Product Type | Two-component one-step multiplex RT-qPCR master mix |
| Function / Efficacy | Enables one-tube reverse transcription and multiplex probe-based qPCR with carryover control and rapid cycling options. |
For research use only, not for clinical use.
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