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Description
RT-qPCR master mix designed to prevent carry-over qPCR contamination making it ideal for use in high-throughput laboratories.
SensiFAST™ Probe dUTP Lo-ROX Mix is a fast, highly reproducible real-time PCR master mix that has been designed with a highly optimized buffer chemistry and hot-start DNA polymerase, to deliver excellent results and very low background, allowing greater sensitivity.
Product Highlights
Optimized: dTTP to dUTP ratio optimized for high sensitivity and specificity
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
Reproducible: increased consistency between technical replicates for increased confidence in results
Fast: delivers reproducible, multiplex accurate assay results in as little as 30 minutes
Product Description
SensiFAST™ Probe dUTP Lo-ROX Mix is a 2x, combination of the latest advances in buffer chemistry and PCR enhancers and stabilizers, together with an antibody-mediated hot-start polymerase, dNTPs and MgCl2. However, some of the dTTPs in the dNTP mix has been substituted for dUTP, so that Uracil DNA Glycosylase (UDGase) can be added to remove carry-over qPCR contamination.
The advanced buffer chemistry and enhancers has been developed for fast qPCR and is designed for superior sensitivity and specificity with probe‑detection technology, including TaqMan®, Scorpions® and molecular beacon probes, and has been validated on all commonly used real-time instruments that require a low concentration of the passive reference dye ROX, making SensiFAST Probe dUTP Lo-ROX Mix perfect for multiplexing, allowing more samples to be run in a day with the highest confidence, ideal for high‑throughput assays.
Applications
Gene expression analysis
Viral and bacterial detection
GMO testing
SNP genotyping
Mutation detection
Environmental monitoring
Fig. 1 Sensitivity and reproducibility
A 10-fold serial dilution of human DNA (in quadruplicate) over five orders of magnitude amplified using SensiFAST Probe dUTP Mix. The results illustrate that SensiFAST Probe dUTP Mix gives highly sensitive and reproducible results.
Fig. 2 Multiplexing
A 10-fold serial dilution of human cDNA amplified with four different probes (only one is shown here) in quadruplex using SensiFAST Probe dUTP Mix (red) and a Kit from supplier T (black) using the manufacturers recommended protocol. The results illustrate that SensiFAST Probe dUTP Mix is more sensitive than supplier T in a multiplex reaction.
Introduction to SensiFAST
Overview, features and benefits of the SensiFAST product family
BIO-84201: 200 x 20 µL Reactions: 5 x 1 mL
BIO-84220: 1000 x 20 µL Reactions: 4 x 5 mL
Storage & Stability
All kit components should be stored at -20°C upon receipt. When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date on the outer box label.
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 recommended PCR conditions in the manual refer to the set-up of TaqMan probe-based PCR. If you want to use other probe types, it may be necessary to adapt the PCR setup conditions, please refer to the relevant literature. In case of hybridization probes it would be necessary to switch to a three-step PCR setup with annealing at 60°C and extension at 72°C and acquire the data at the end of the annealing phase (the exact annealing temperature may vary and has to be optimized for a specific assay). The two-step protocol with an annealing / extension step at 60°C would lead to the degradation of these probes, which is wanted for TaqMan but not for hybridization probes.
If you have further questions, please contact Meridian Technical Support.
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).
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.
