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What is the excitation source for the Mx3000P™ system*? The system’s excitation source is a quartz tungsten halogen lamp, which emits light in the range of 350 to 750 nm. The bulb comes in a pre-aligned lamp fixture for easy replacement without system downtime or the need for a service call. How is the emitted fluorescence detected? The Mx3000P system utilizes a photomultiplier tube (PMT) to detect fluorescence. This PMT design provides the most sensitive detection technology used in real-time instrumentation. What filter sets are available for the Mx3000P system? You can choose the four excitation and detection filter sets to be installed on your Mx3000P system. Our standard filter sets include (1) FAM™/SYBR® Green I (492 nm ex. – 516 nm em.), (2) HEX™/JOE™/VIC™ (535 nm ex. –555 nm em.), (3) ROX™/Texas Red® (585 nm ex. 610 nm em.), and (4) Cy5™ (635 nm ex. – 665 nm em.). We also offer ALEXA Fluor® 350 (350 nm ex. – 440nm em.), TET (517 nm ex. – 538 nm em.), Cy3™ (545 nm ex. – 568 nm em.), and TAMRA™ (556 nm ex. – 580 nm em.) filters, which can be substituted for any of the standard filter sets. What temperature uniformity and ramp rate is the Mx3000P thermal system able to achieve? The Mx3000P system provides thermal uniformity of +/- 0.25 degrees across the 96-well thermal block. The ramp rate is up to 2.5oC per second and is dynamically controlled by the software to ensure optimal results. What level of sensitivity and range of detection does the Mx3000P system provide? The system routinely achieves a linear dynamic range of seven orders of magnitude using a plasmid template target (5 to 5 x 107 copies). Is there any cross-talk between different fluorophores in a multiplex assay on the Mx3000P system? The precision design of the Mx3000P optical system blocks cross-talk levels between fluorophores to less than 0.5%. What kind of service and support can I expect after purchasing the Mx3000P system? Stratagene provides a variety of support and training options to help you get started in your real-time PCR research. Immediately after purchasing your instrument, we will send you an introductory guide, Introduction to QPCR Methods, which discusses the basics of real-time PCR technology, real-time applications, and a diagram of the Mx3000P system. We also offer recurring web-based QPCR seminars available at several convenient times to match your schedule and time zone. The seminar topics include quantitative methods using real-time PCR, probe and primer design and optimization, Mx3000P software training, and multiplex experiment design. Seminars covering advanced QPCR topics are also scheduled. Please check the Mx3000P system website (www.mx3000p.com) for a list of online seminars and registration instructions. |
When the Mx3000P system arrives in your laboratory, you will receive a validation test plate that allows you to quickly qualify the system and get started right away. A voucher for a regional training clinic is included with each Mx3000P system. This training program provides similar topics to the web-based training, with the advantage of individual instruction and data analysis provided by our QPCR applications scientists. In addition, we have a growing collection of application notes and research publications by scientists using the Mx3000P system and other real-time PCR products from Stratagene. The Mx3000P instrument has moving parts, like the scanning arm mechanism, which are not found in some other real-time systems. Should I be concerned about reliability? Reliability is always important when evaluating your instrument options. The design of the Mx3000P system is simple and robust, using proven industry technology. The scanning arm mechanism is simply a motor and pulley drive belt. We use the same design in our Mx4000® multiplex QPCR system. This is also the same kind of reliable, long-standing mechanical system used with the print head in inkjet printers. I’ve been told that I need a gradient function to optimize my primers. Does the Mx3000P thermal system provide a gradient function? The method of using a thermal gradient to optimize primers for PCR was developed by Stratagene with the introduction of the RoboCycler® temperature cycler in 1994. In standard PCR, this feature allows a researcher to determine the optimal annealing temperature within a single run. While a gradient can be used in a similar fashion for QPCR optimization, it produces suboptimal results. A gradient method requires using a fixed primer concentration. The melting temperature selected is a compromise between the optimal temperatures for each primer set when they are run together. This compromise in the reaction conditions can result in sub-optimal reaction kinetics and means that each experiment will be run under different conditions. The preferred method for QPCR assay design, and the method used on the Mx3000P system, maintains constant annealing temperature for all primer sets. Primer sets are optimized by titrating the primer concentration for optimal PCR kinetics. All primer sets for every experiment are run with the same thermal profile, allowing analysis of many genes using uniform reaction conditions in the same experiment. Because all reactions are designed using the same conditions, this method has the additional benefit of allowing an easier transition to multiplex experiments. If TaqMan® chemistry is being used, the gradient optimization method tends to be less useful because the choice of temperatures for your twostep PCR is dependent not only on the primer and probe Tms, but also on the temperature at which Taq has the proper 5’ to 3’ exonuclease activity. The Mx3000P system takes about 18 seconds to excite and read each of the 96 wells when all four filters are being used. Is the real-time data uniform and reproducible given the time it takes to read all 96 wells? Using empirical testing, Stratagene has proven that this scanning method does not affect quantitation uniformity results. The time delay between reading the first well and the last well will not affect the Cts recorded. As a default, three scans are completed and averaged during each time point to provide the most uniform and reproducible fluorescence data (Ct) results.
For more information about the Mx3000P system and Stratagene’s QPCR product portfolio, please visit www.stratagene.com/qpcr. |
