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Description
Outstanding assay reproducibility, sensitivity and robustness from both DNA and RNA templates, under fast thermal cycling conditions.
Product Highlights
Reproducible – consistent results between technical replicates for increased confidence in results
Specific – antibody-mediated hot-start DNA polymerase minimizes non-specific amplification for improved assay sensitivity and reliability
Sensitive – reliable quantification of low abundance targets and scarce samples
Robust – reliable, accurate detection of DNA and RNA targets from a broad range of sample types
Fast – delivers reproducible, accurate assay results in as little as 30 minutes
Product Description
The SensiFAST™ SYBR & Fluorescein Kit has been developed for fast, 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 combination of the latest advances in buffer chemistry and PCR enhancers ensures that the SensiFAST SYBR & Fluorescein Kit produces reliable assay results under fast thermal cycling conditions. An antibody-mediated hot-start DNA polymerase system promotes highly-specific amplification, in turn improving assay sensitivity and dynamic range.
The SensiFAST 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 / RNA target detection
miRNA profiling / quantification
Copy number variation (CNV) analysis
Fig. 1 SensiFAST SYBR using fast cycling conditions
A fragment of γ-actin was amplified using SensiFAST SYBR & Fluorescein (red) and the results were compared with amplification using a kit from Supplier I (blue). The process using a 10 fold serial dilution of human genomic DNA over four orders of magnitude. The results illustrate that SensiFAST SYBR & Fluorescein is as faster (earlier Ct) and more efficient than Supplier I.
Fig. 2 Sensitivity of SensiFAST SYBR & Fluorescein
A fragment of ubiquitin gene was amplified using SensiFAST SYBR & Fluorescein (red) and the results were compared with amplifications using a Kit from supplier Q (blue). The process used a 10 fold serial dilution of human RNA (in triplicate) over five orders of magnitude. The results illustrate that a) SensiFAST SYBR & Fluorescein was faster (earlier Ct) and more sensitive than supplier Q and b) there was far less primer-dimer formation.
A discussion of the pros and cons of each detection strategy.
I had a very good experience with the SensiFAST SYBR. It was quick and easy to use, while also providing me with near perfect R-squared values and dissociation curves. Even when compared to other, more expensive, master mixes, I obtained results of equal or better quality.
Mariah Meyer, University of Montana, DBS, US
Instrument Compatibility
Please refer to the Real-Time PCR Selection Chart to confirm the SensiFAST SYBR® & Fluorescein Kit is compatible with your instrument.
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.
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
""I started my real-time PCR analyses with SensiMix SYBR & ...
"
CSIRO Plant Industry, Canberra, Australia
"I started my real-time PCR analyses with SensiMix SYBR & Fluorescein and found it very effective, with many primer pairs amplifying with close to 100% efficiency. When it became available, I was able to switch to the SensiFAST SYBR & Fluorescein without compromising the results, resulting in dramatically shorter cycling times."
Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments (see Product Selection Tool), the SensiFAST SYBR & Fluorescein contains fluorescein premixed in the mastermix at optimised concentrations.
Although SensiFAST kits have been designed for fast PCR on the new generation of fast machines, they will work equally well for standard or fast PCR protocols on all real-time PCR machines.
Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95°C for 2-3 minutes.
When comparing SensiFAST 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.
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).
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.
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.
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.
