RT² SYBR® Green qPCR Mastermix

Description

Protocol

Components

The RT² SYBR® Green qPCR Mastermix contain all of the optimized reagents and buffers needed for SYBR® Green based real-time polymerase chain reactions in all real-time PCR instrumentation including ABI® Bio-Rad® Stratagene® Roche, and Eppendorf® Each Mastermix includes real-time PCR buffer, a high-performance HotStart DNA Taq polymerase, nucleotides, and ROX® or Fluorescein reference dye if and when needed to normalize a specific instruments' optics. Simply add the Mastermix to PCR tubes along with your template and primers. The chemically-modified and tightly controlled HotStart enzyme uniquely provides more accurate SYBR® Green results by preventing the amplification of primer dimers and other non-specific products. These RT² SYBR® Green qPCR Mastermix are best suited for real-time PCR applications using SYBR® Green based detection like the RT² Profiler™, RT² miRNA, EpiTectChIP and EpiTectMethyl DNA Methylation PCR Arrays and qPCR Assays from SABiosciences. RT² SYBR® Green qPCR Mastermix, from the experts in high-performance quantitative PCR, provide: Accuracy: Guaranteed single gene-specific products with minimal primer dimers Reliability: Maximized amplification efficiencies Convenience: Matched to the instrument in your lab with specially formulated all-in-one mixes

Description

Protocol

Components

Ensure that you do not contaminate the 2X real-time PCR cocktail by dividing into aliquots containing the amount of cocktail necessary for the number of reactions you are preparing each day. The rest of the cocktail should be kept in storage away from any sources of template DNA. When using RT² qPCR Mastermix with RT² Profiler™ PCR Arrays, RT² qPCR Primer Assays, EpiTectMethyl PCR Arrays, DNA Methylation qPCR Assays, RT² miRNA PCR Arrays, RT² miRNA qPCR Assays, or EpiTectChIP PCR Arrays & Real-time ChIP-qPCR Primer Assays, refer to the appropriate User Manuals for reaction mixtures. For general PCR purposes, mix the following components in a PCR tube: 12.5 µl RT² SYBR® Green qPCR Mastermix 10.5 µl ddH2O 1.0 µl template cDNA (up to 250 ng) 1.0 µl gene-specific 10 µM PCR primer pair stock 25.0 µl final volume Recommended real-time thermal cycler program. Please find your product for the correct cycling conditions. *NOTE: The 10 min step at 95 ºC is required to activate the HotStart Taq DNA polymerase. RT² Profiler PCR Arrays & RT² Primer Assays Use Two-Step Cycling Program For: BioRad® iCycler, MyiQ® and iQ™5.      95ºC, 10 min; 40 cycles of (95ºC, 15 sec; and 60ºC, 60 sec) Use Three-Step Cycling Program For: All Other Instruments      95ºC, 10 min; 40 cycles of (95ºC, 15 sec; 55ºC, 30-40 sec, and 72ºC, 30 sec) For the following products, please refer to the corresponding user manuals for the correct cycling programs: EpiTectMethyl PCR Arrays & DNA Methylation qPCR Assays RT² miRNA PCR Arrays & miRNA qPCR Assays EpiTectChIP PCR Arrays & Real-time ChIP-PCR Primers Program the real-time thermal cycler to detect and record the SYBR?Green signal from every reaction at the end of the 60ºC annealing / extension step of each cycle. Run your instrument's default melting curve program immediately after the above PCR program, and generate a first derivative dissociation curve. No more than one peak should appear in each reaction If your instrument does not have a default melting curve program, run the following program instead: 95ºC, 1 min; 65ºC, 2 min (OPTICS OFF); 65ºC to 95ºC at 2ºC/ min (OPTICS ON). NOTE: Be sure to visually inspect the plate after the run for any signs of evaporation from any of the wells. If evaporation is observed, make a note of which wells so that you may qualify your data analysis appropriately. Back to Top

Description

Protocol

Components

Please check the kit components immediately after you receive this package. We are not responsible for any missing items not reported within two (2) business days upon receipt. Contents: Catalog #  Contents 330510 Two (2) tubes each containing 1.35 mL of 2X solution and enough for 100 standard 25-µl reactions 330512 Twelve (12) tubes each containing 1.35 mL of 2X solution and enough for one PCR Array 330513 Twenty-four (24) tubes each containing 1.35 mL of 2X solution and enough for one PCR Array 330511 Eight (8) tubes each containing 1.35 mL of 2X solution and enough for 4 384-well PCR Arrays 330519 One (1) bottle containing 25 ml of 2X solution and enough for 2000 standard 25-µl reactions Storage Conditions: The kit is shipped on dry ice or cold packs. For long-term storage, keep at -20 ºC. If entire volume is not be used all at once, divide into aliquots and store at -20 ºC. Avoid repeated freezing and thawing. Shelf Life: All reagents are stable for 6 months after receipt if stored at the recommended temperature. SYBR® is a registered trademark of Molecular Probes, Inc.; SmartCycler® is a registered trademark of Cepheid; LightCycler® is a registered trademark of Roche Diagnostics; ABI® is a registered trademark of Applera Corporation; FAM™ is a trademark of Applera Corporation; ROX™ is a trademark of Applera Corporation; Bio-Rad® and iCycler® are registered trademarks of Bio-Rad Laboratories; Chromo4™ iQ™, MyiQ™ and Opticon™ are trademarks of Bio-Rad Laboratories; Stratagene® is a registered trademark of Stratagene Corporation; Eppendorf® and Mastercycler® are registered trademarks of Eppendorf AG; RT² and RT² Profiler™ are trademarks of SABiosciences Corporation. Use of this product is covered by one of more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152 (claims 1 to 23 only), 5,773,258 (claims 1 and 6 only), 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Back to Top