RT² SYBR® Green/Fluorescein FAST Master Mixes

Description

Protocol

Component

The RT² SYBR Green/Fluorescein Fast master mix has been optimized to use a fast PCR protocol, without compromising specificity or sensitivity, using SYBR Green based detection on instrumentation using the Fluorescein reference dye, such as the: BioRad® iCycler®, iQ™5, MyiQ™ The RT² SYBR/Fluorescein Green Fast master mix contains all of the reagents and buffers required for fast real-time polymerase chain reactions on instrumentation requiring the Fluorescein reference dye, including an improved proprietary real-time PCR buffer, a high-performance HotStart DNA Taq polymerase, nucleotides, SYBR Green dye, and the Fluorescein reference dye needed to normalize the instruments' optics. Simply add the master mix to PCR tubes or plates 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.

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. For general real-time PCR purposes, mix the following components in a PCR tube: 12.5 µl RT² SYBR® Green Fast Master Mixes 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: NOTE: The 10 min step at 95 ºC is required to activate the HotStart Taq DNA polymerase.        95 ºC, 10 min; 40 cycles of (95 ºC, 10 sec; 60 ºC, 30 sec). For ABI 7500, select the "Fast 7500" in the Run Mode panel. 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. Please put the appropriate table below with the corresponding selection from above. Catalog Number  Contents 330600 Two (2) tubes, each containing 1.35 mL of 2X solution and enough for 200 25-µl reactions 330602 Twelve (12) tubes, each containing 1.35 mL of 2X solution and enough for 1200 25-µl reactions 330603 Twenty-four (24) tubes, each containing 1.35 mL of 2X solution and enough for 2400 25-µl reactions 330601 Eight (8) tubes, each containing 1.35 mL of 2X solution and enough for 800 25-µl reactions 330609 One (1) bottle containing 25 ml of 2X solution and enough for 2000 25-µl reactions Catalog Number  Contents 330610 Two (2) tubes, each containing 1.35 mL of 2X solution and enough for 200 25-µl reactions 330612 Twelve (12) tubes, each containing 1.35 mL of 2X solution and enough for 1200 25-µl reactions 330613 Twenty-four (24) tubes, each containing 1.35 mL of 2X solution and enough for 2400 25-µl reactions 330611 Eight (8) tubes, each containing 1.35 mL of 2X solution and enough for 800 25-µl reactions 330619 One (1) bottle containing 25 ml of 2X solution and enough for 2000 25-µl reactions Catalog Number  Contents 330620 Two (2) tubes, each containing 1.35 mL of 2X solution and enough for 200 25-µl reactions 330622 Twelve (12) tubes, each containing 1.35 mL of 2X solution and enough for 1200 25-µl reactions 330623 Twenty-four (24) tubes, each containing 1.35 mL of 2X solution and enough for 2400 25-µl reactions 330621 Eight (8) tubes, each containing 1.35 mL of 2X solution and enough for 800 25-µl reactions 330629 One (1) bottle containing 25 ml of 2X solution and enough for 2000 25-µl reactions Storage Conditions The kit is shipped on dry ice or cold packs. For long-term storage, keep at -20ºC. If entire volume is not be used all at once, divide into aliquots and store at -20ºC. Avoid repeated freezing and thawing. Shelf Life All reagents are stable for 6 months after receipt if stored at the recommended temperature. NOTICE TO PURCHASER: LIMITED LICENSE 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