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Description
Outstanding assay reproducibility and sensitivity for both DNA and cDNA templates, under standard conditions.
Product Highlights
Sensitive - reproducible detection of low-copy number templates
Reproducible – consistent results between technical replicates for increased confidence in results
Specific - proprietary hot-start modification minimizes non-specific amplification for improved assay reliability in high-throughput assays
Robust – reliable, accurate detection of DNA and cDNA targets from a broad range of sample types
Product Description
The SensiMix™ SYBR® & Fluorescein Kit has been developed for highly accurate real-time PCR and has been validated on real-time PCR platforms that require the passive reference dye fluorescein for collection of dynamic well factors.
A proprietary, highly-optimized, chemical hot-start PCR enzyme promotes highly-specific amplification, in turn improving assay sensitivity and dynamic range, ensuring that SensiMix SYBR® & Fluorescein Kit provide the sensitivity and specificity required for demanding assays under standard thermal cycling conditions.
The SensiMix SYBR® & Fluorescein Kit has been optimized to deliver optimal performance in tandem with the SensiFAST cDNA Synthesis Kit, which offers fast, unbiased cDNA synthesis, without compromising cDNA yield or coverage.
Applications
Gene expression analysis
DNA / cDNA target detection
miRNA profiling / quantification
Copy number variation (CNV) analysis
Broad dynamic range SensiMix SYBR & Fluorescein Kit
Amplification curves were obtained for the YWHAZ gene over 5 log dilution series of mouse cDNA. Reactions were in quadruplicate, using standard reaction conditions. The results demonstrate even spread of the curves with no inhibition even at the highest cDNA inputs.
Reduced primer dimer using the SensiMix SYBR & Fluorescein Kit.
Melt curve analysis were obtained for the YWHAZ gene over 5 log dilution series of mouse cDNA. The presence of one peak at 82°C indicates that the fluorescence is not attributable to primer-dimers, even in the presence of low template concentrations of template, demonstrating that the kit consistently provides high specificity.
All kit components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided. Avoid exposure of the SYBR® Green I to light.
When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date indicated on the outer box label.
The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR®) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.
The amount of the ROX passive reference dye needed varies depending on the instrument optics. Our SensiFAST kits have been optimized for all these different instruments (see Product Selection Tool).
Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments (see Product Selection Tool), the SensiMix SYBR® & Fluorescein contains fluorescein premixed in the mastermix at optimized concentrations.
Multiple products in melting curve analysis may be caused by unspecific products or primer dimer. However, appearance of multiple bands in melting curve analysis does not necessarily show different products. Multiple peaks may also appear, if distinct regions of the PCR product melt at different temperatures (e.g. due to an inconsistent GC content). We recommend to analyze the PCR product on a gel, to decide if unspecific products are present. The troubleshooting guide of your product insert includes several suggestions to solve these problems. In many cases problems with non-specific products can be solved if the annealing temperature is increased and/or the elongation time is decreased.
If you have further questions, please contact Meridian Technical Support.
SYBR® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in real-time. This dye is very attractive for researchers analysing lots of different genes.
The probe system is always specific, probes only detect the gene of interest. It is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimization than SYBR® Green I assays.
Yes, polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase SensiMix contains a chemical hot-start that is only activated after heating at 95°C for 10 minutes, making SensiMix very stable at room temperature..
This method is only effective if all the researchers in the laboratory, or using the thermocycler, are all using a dUTP/dNTP and UNG system. If even one researcher is not, it ceases to be an effective control. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency. Most labs do not need to use the dUTP method of control, optimised protocols will allow high specificity of PCR and good lab practices (using disposable consumables, the use of filter tips and maintaining a separate area for PCR set-up and PCR amplification and any post-PCR analysis) virtually eliminate the risk of cross contamination.
In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy in handling, making it ideal for high throughput screening.
In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Meridian sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.
When comparing SensiMix with a mix from another supplier we strongly recommend to use the suggested conditions for PCR setup and cycling. Every real-time mix has its specific composition and different properties. That’s why they should be tested with their specific conditions, mostly this is possible on different plates only. We recommend amplifying from a ten-fold template dilution series. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed.
A possible reason is that the template amount is too concentrated. An early Ct may impair the baseline correction and may cause these strange curves. We suggest to dilute the template. Alternatively if the curve is flattened, this may be due to inhibitors in the reaction. Running a serial dilution of your sample will dilute the inhibitor concentration, if this corrects the problem, you will either need to go back and re-purify your template, or use it at a lower concentration, where the inhibitor effect has been diluted out.
