Hotstart HiTaq& Super M-MuLV One Step RT-qPCR Kit (with UDG) is widely recognized for its exceptional speed and quantitative accuracy. This product enables continuous Real-Time RT-PCR reactions within a single tube, simplifying operations. The enzyme system employs thermolabile UDG (Uracil-DNA Glycosylase) in combination with dUTP-containing substrates, effectively preventing carryover contamination from previous PCR products. The thermolabile UDG is completely inactivated at 50°C, ensuring it does not interfere with the reverse transcription products of the current reaction.
This kit utilizes a highly stable and efficient reverse transcriptase (Super M-MuLV Reverse Transcriptase) and a chemically modified hot-start DNA polymerase (Hotstart HiTaq DNA Polymerase). It delivers high amplification efficiency and specificity, enabling robust Real-Time One-Step RT-PCR. This significantly enhances detection sensitivity and eliminates the need for post-PCR electrophoresis, making it particularly suitable for the detection of trace amounts of RNA.
Specifically designed for convenient and highly sensitive one-step RT-PCR, this kit features a unique enzyme formulation and specially optimized buffer system. These ensure efficient and accurate reverse transcription and PCR amplification without the need for reaction optimization. It generates excellent standard curves over a wide quantitative range, allowing precise quantitative detection of target genes with high reproducibility and reliability.
Applications: Quantitative or qualitative detection of nucleic acids using RNA as template. Reverse transcription and amplification are completed in a single tube without tube opening.
Product Component List
FP1509101 | Components | 100T | 1000T | 10000T | Storage |
FP1509101A | Hotstart HiTaq& Super M-MuLV Mix | 200 μL | 2×1.0 mL | 20 mL | -20℃ |
FP1509101B | Buffer Mix | 950 μL | 10×0.95 mL | 95 mL | -20℃ |
Protocol:
1. Reaction Setup:
Thaw and thoroughly mix all PCR solutions required for the reaction.
Briefly centrifuge the solutions and keep them at room temperature until use.
Probes should be protected from light.
Recommendation: Aliquot PCR reagents to avoid repeated freeze-thaw cycles.
2. Prepare the PCR Reaction Mixture according to the table below:
Component
| Volume/25 μL
| Volume/50 μL
| Final Concentration
|
Buffer Mix
| 9.5 μL
| 19 μL
| 1×
|
Enzyme Mix (Hotstart HiTaq& Super M-MuLV)
| 2 μL
| 4 μL
| 2/50 μL
|
Primer-Probe Mix
| 1 μL
| 2 μL
| / |
Template
| 5 μL
| 10 μL
| /
|
ddH₂O | To 25 μL
| To 50 μL
| /
|
Total Volume
| 25 μL
| 50 μL
|
|
Note: The amounts of primers, probes, and template can be adjusted according to experimental requirements.
3. Mixing:
Gently mix the reaction components by pipetting up and down or by brief vortexing. Centrifuge briefly at room temperature to collect the liquid at the bottom of the tube.
4. PCR Amplification:
Place the PCR reaction tubes in the thermal cycler and initiate the PCR program.
Standard Thermal Cycling Protocol:
Step
| Temperature
| Time
| Cycles
|
Reverse Transcription
| 50℃
| 15min
| 1 |
Initial Denaturation
| 95℃
| 10min
| 1 |
Denaturation
| 95℃
| 10s
| 45 |
Annealing/Extension
| 60℃
| 40s
| 45
|
Hold
| 12℃
| ∞
| 1 |
Note: Reaction conditions can be adjusted based on specific experimental requirements.
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