KAPA SYBR FAST One-Step qRT-PCR Kits are designed for optimal performance in stringent qRT-PCR reaction conditions, exhibiting dramatic improvements in sensitivity, specificity, reaction efficiency, and fluorescence. The kit is optimized for rapid one-step, one-tube RNA quantification, reducing experimental variation and contamination with a convenient qRT-PCR protocol.
KAPA SYBR FAST One-Step qRT-PCR Kits contain M-MuLV reverse transcriptase, RNase inhibitor, and a novel DNA polymerase evolved via directed evolution.
KAPA SYBR® FAST One-Step is suitable for:
- One-step quantitative reverse transcriptase PCR (qRT-PCR)
- Gene expression analysis
- Low-copy gene detection
- Microarray validation
- miRNA research
- Gene knockdown validation
KAPA SYBR® FAST DNA Polymerase displays no enzymatic activity at ambient temperature. This prevents the formation of misprimed products and primer-dimers during reaction setup prior to the first denaturation step and results in high PCR specificity and accurate quantification. Moloney murine leukemia virus reverse transcriptase (M-MuLV-RT) has a high affinity for RNA and is optimized for cDNA synthesis at 42°C. The RNase Inhibitor safeguards against degradation of RNA target due to RNase contamination.
Features and Benefits
Increase signal and reaction efficiency:
- Consistently higher amplification efficiencies for accurate qPCR
- Higher fluorescence, earlier Cq values and improved reaction efficiencies
Amplify across a broad range of target lengths:
- Robust performance independent of amplicon size
Improve sensitivity and reproducibility:
- Accurate interrogation across a wide range of RNA template concentrations
- This kit contains wild-type M-MuLV and an engineered enzyme optimized for qPCR using SYBR Green I dye chemistry.
- The 2X master mix contains a proprietary buffer. Together with the novel enzyme, this improves amplification efficiency of both GC- and AT-rich targets.
- Use only gene-specific primers for one-step qRT-PCR.
- Optimal cDNA synthesis is achieved at 42°C for 5 min.
- 3 min at 95°C is sufficient for RT inactivation and DNA polymerase activation.
- For 3-step cycling, use 20 sec for primer annealing and 1 sec for extension/data acquisition at 72°C.
- Do not exceed 25 μL reaction volumes.
All kit components are subjected to stringent functional quality control, are free of detectable contaminating exo and endonuclease activity, and meet strict requirements with respect to DNA contamination.
Always ensure that components have been fully thawed and thoroughly mixed before use. The KAPA SYBR FAST qPCR Master Mix (2X) may not freeze solidly, even when stored at -20°C. The KAPA RT Mix is temperaturesensitive, and should be stored at -20°C and kept on ice during use.
The SYBR Green I dye contained in the KAPA SYBR FAST qPCR Master Mix (2X) and ROX/fluorescein dyes (depending on kit configuration) are light sensitive. Exposure to direct light for an extended period of time will result in loss of fluorescent signal intensity.
KAPA SYBR FAST qPCR Master Mix (2X) is stable through 30 freeze-thaw cycles. Ensure that all reagents are stored protected from light at -20°C when not in use. When protected from light, reagents are stable in the dark at 4°C for at least one week and may be stored at this temperature for short-term use provided that they do not become contaminated with microbes and/or nucleases.
For Research Use Only. Not for use in diagnostic procedures.
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