2X One-Step Probe RT-qPCR Master Mix
Croyez 2X One-Step Probe RT-qPCR Master Mix is a one-step real-time reverse transcription-polymerase chain reaction (RT-qPCR) kit developed for cDNA synthesis and real-time PCR in the same tube. This product is suitable for probe-based detection and formulated as a 2-fold premix. Reaction can be simply set up by adding the RNA template, primers, and probes. This master mix does not contain ROX reference dye; it offers great convenience and minimizes the risk of cross-contamination.
The following procedure is a general guideline for One-step RT-qPCR reaction. To maintain an RNase-free environment, always wear disposable gloves, and use laboratory consumables and water of nuclease-free grade during the whole experiment course.
RT-qPCR reaction set-up:
1. Place all required reagents on ice.
* See Usage Notes for additional guidelines on primer/template preparation.
2. Gently mix the reaction thoroughly to achieve uniform distribution and briefly centrifuge.
3. Thermal cycling conditions for standard qPCR
RT-qPCR reaction set-up:
1. Place all required reagents on ice.
| Component | Amount | Final concentration |
| 2X One-Step Probe RT-qPCR Master Mix | 10 μL | 1X |
| Forward primer (10 μM) | 0.8 μL | 0.4 μL |
| Reverse primer (10 μM) | 0.8 μL | 0.4 μL |
| Probe (10 μM) | 0.4 μL | 0.2 μL |
| RNA template | X μL | ≦1 μg (total RNA) |
| Nuclease-Free H2O | Y μL | - |
| Total reaction volume | 20 μL | - |
2. Gently mix the reaction thoroughly to achieve uniform distribution and briefly centrifuge.
3. Thermal cycling conditions for standard qPCR
| Step | Cycles | Temperature | Time |
| Reverse transcription | 1 | 50°C | 10–15 min |
| Enzyme activation | 1 | 95°C | 5 min |
| Denaturation | 40-45 | 95°C | 5–15 sec |
| Annealing/Extension | 55-65°C | 30–60 sec |