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FluoCycle II™ SYBR® Master Mix 2X Master Mix for Real Time PCR with SYBR® Green Cat# Format Description ERD002100BIM 100 reactions FluoCycle II™ SYBR® Master Mix ERD002250BIM 250 reactions FluoCycle II™ SYBR® Master Mix STORAGE AND STABILITY 1 year at -20°C. The Master Mix tube must be protected from light. KIT CONTENT FluoCycle™ II SYBR® Master Mix is a qPCR mix that may be used with any appropriately designed set of primers to detect any DNA or cDNA sequence. FluoCycle™ II SYBR® Master Mix is a 2X amplification mix for Real Time PCR protocols. The detection of PCR product is based on the binding of the fluorescent dye SYBR®-Green to the double-stranded DNA and fluorescence increases with the amplified PCR product. The kit includes enough reagents to perform either 100 or 250 x 50 μl reactions. FluoCycle™ II Master Mix SYBR® contains a Hot Start Taq DNA polymerase that can be activated by an initial step of 5 minutes at 95°C. Reagent Size FluoCycle™ II SYBR® Master Mix* 1.25 ml (Amber tube) Quantity Formulation 2 vials (100 reactions x 50 μl) 100mM KCl, 20mM Tris HCl pH 8.3, 0.02% Tween-20, 0.8mM of each dNTPs (dATP, dCTP, dGTP, dTTP), 200 units/ml Taq DNA polymerase, 8 mM MgCl2, SYBR® Green I and stabilizers. 5 vials (250 reactions x 50 μl) 100mM KCl, 20mM Tris HCl pH 8.3, 0.02% Tween-20, 0.8mM of each dNTPs (dATP, dCTP, dGTP, dTTP), 200 units/ml Taq DNA polymerase, 8 mM MgCl2, SYBR® Green I and stabilizers. *store the tube away from light NOTES • SYBR® Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR® Green is a dye binding the Minor Groove of double stranded DNA. When SYBR® Green dye binds to double stranded DNA, the intensity of the fluorescent emissions increases. As more double stranded amplicons are produced, the SYBR® Green dye signal will increase. • Primers and probes should be designed within a region including a G/C content ranging from 30% to 80%. Regions with G/C content higher than 80% might be not completely denatured during thermal cycling, causing lower amplification yield. G/C-rich sequences are also prone to nonspecific interactions decreasing the amplification efficiency and producing a non-specific signal in SYBR® Green dye assays. For the same reason, primers containing sequences of four or more G/C bases should be avoided. • To avoid non-specific annealing, primers should not contain more than two C/G repetitions at the last five positions of their 3’ end. Maintaining the amplified fragment shorter than 150bp while satisfying this requirement might be difficult in case of highly G/C content. In general try to design primers with the lowest content of G/C repetitions at the 3’ end. • The primers concentration should be optimised through an initial range from 50nM to 300nM. Because the individual efficiency of the forward and reverse primer may vary, their respective concentrations must vary to compensate. Therefore all permutations of a selected number of primer concentrations must be tested. For instance, there are four possibilities of how forward and reverse primer concentrations could be combined for the following individual concentrations: 50nM, 300nM: Forward/Reverse: 50/50, 50/300, 300/50, 300/300. APPLICATION PROTOCOL We recommend to prepare a PCR mix considering total n. + 1 samples. Add all reagents in a sterile tube and transfer the right volume of the resulting solution in the final reaction plate or tubes. Add the appropriate volume of DNA template. The reagents of PCR mix have to be mixed under the following ratio (see table 1 below): Table 1: reaction set up Reaction Component Volume Final conc. – total quantity FluoCycle™ II SYBR Master Mix 25 μl 1X Primers Forward variable 50-300 nM Primers Reverse variable 50-300 nM DNA template variable 20 ng - 100 ng Sterile Water to reach 50 μl NA Total reaction volume 50 μl ® Transfer the tubes/plate inside the Real Time PCR Platform and run the following program of amplification: SUGGESTED THERMAL PROFILE Time Temperature Cycles 5 min 95°C 1 15 sec 95°C 45 60 sec* 60°C* Fluorescent Acquisition *The exact values for time and temperature must be calculated according to the primers sequences and the amplicon length SET MELTING CURVE From 65°C to 95°C increasing of 0.2°C each time Fluorescence ON NOTICE TO PURCHASER: LIMITED LICENSE For Research Use Only (RUO). Diagnostic uses under Roche patents require a separate license from Roche. SYBR® is a registered trademark of Molecular Probes, Inc. No right under any patent claim (ex: Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial servicesof any kind, including without limitation reporting the results of purchaser’s activitiesfor a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. Some applications this product is used in may require a license which is not provided by the purchase of this product. Users should obtain the license if required. Further information on purchasing licenses: Director of Licensing, Applied Biosystems, 850 Lincoln CentreDrive, Foster City, California 94404 USA. MOLECULAR BIOLOGY EuroClone S.p.A. Via Figino 20/22, 20016 Pero (MI) - Italy Phone: +39.02.38195.1 - Fax +39.02.38101465 e-mail: [email protected] www.euroclone.it I-ERD002_ed0/0813/693_FluoCycleII SYBR The fluorescence signal registered by the instrument detects the presence of the DNA. Binding of SYBR® Green I to double- stranded DNA is nonspecific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the need for agarose gel electrophoresis because the melting temperature (Tm) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SYBR® Green I for real-time PCR multiplex reactions, discrimination of amplicons could be possible, provided the Tm values are sufficiently different.