QUICK REFERENCE
SYBR® Select Master Mix
Cat. nos. 4472903, 4472908, 4472918, 4472919, 4472920, 4472913
Publication Part no. 4473368 Rev. Date 8 May 2013 (Rev C) This quick reference card provides simplified instructions for using the SYBR® Select Master Mix reagent for performing real-
time PCR assays. For detailed instructions and ordering information for additional products, refer to the SYBR® Select Master Mix Ur Guide available at /manuals.
Performing Real-Time PCR Assays
General Guidelines
• Prepare single-stranded cDNA from total RNA or mRNA samples. For optimal performance, u up to 100 ng of cDNA for
10 µL or 20 µL reactions.
• Mix the SYBR® Select Master Mix thoroughly before u by swirling the bottle.
• For best results, it is recommended to perform four replicates of each reaction.
• Master mixes can be prepared depending upon your experimental requirements. Scale the components to be included in the master mix according to the number of reactions to be performed. Include an additional 10% of the master mix volume to account for variations in pipetting.
• If using smaller reaction volumes, scale all components of the reaction mix proportionally. Reaction volumes <10 µL are not recommended.
CHEMICAL HAZARD. SYBR® Select Master Mix (2X) may cau eye, skin and respiratory tract irritation. Read the Safety Data Sheet (SDS), and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. For safety and biohazard guidelines, refer to the Safety ction in the SYBR® Select Master Mix Protocol.
Prepare the PCR Reactions
Prepare the appropriate number of reactions according to the volumes in the following table:
1.
StepOnePlus® Real-Time PCR Systems, u≤200 nM of each primer. For optimal performance in in Fast mode (384-well plates), increa primer concentration toward the higher end of the recommended range (e.g. 400 nM). If using StepOne® or StepOnePlus® Real-Time PCR Systems in Fast mode, u 300–400 nM of each primer.
2. Mix the components thorougly, then centrifuge briefly to spin down the contents and eliminate any air bubbles.
3. Transfer the appropriate volume of each reaction to each well of an optical plate.
4. Seal the plate with an optical adhesive cover, then centrifuge the plate briefly to spin down the contents and eliminate any
air bubbles.
Note:PCR can be performed on the reaction plate at any time up to 72 hours after completing the reaction tup when kept at room temperature, and protected from exposure to direct light.
For rearch u only. Not for u in diagnostic procedures.
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Run the PCR Reaction Plate
Run the plate on a Life Technologies real-time quantitative PCR instrument. See the appropriate instrument ur guide for help with programming the thermal-cycling conditions or with running the plate. To run the plate:
1. Place the reaction plate in the instrument.
2. Set the thermal cycling conditions using the default PCR thermal-cycling conditions specified in the following tables
according to the melting temperature of your primers:
™™** Denature for 3 conds when using 7500 Fast, StepOne ®, or StepOnePlus ® Real-Time PCR Systems.
3. Set the instrument to perform a default dissociation step. Note : A dissociation curve can be performed up to 72 hours after
the real-time PCR run if the plate is stored in the dark, or up to 24 hours later if the plate is stored expod to light.
4. Set the reaction volume appropriate for the type of plate being ud for your PCR reaction.
5. Start the run.
Analyze Your Results
• View the amplification plots.
• Adjust the baline and threshold values to determine the threshold cycles (C T ) for the amplification curves. • U the standard curve method or the relative quantification (∆∆C T ) method to analyze results.
• When performing melt curves with StepOne ®, or StepOnePlus ® instruments, delect the passive reference (e manual for details).
NOTICE TO PURCHASER: PLEASE REFER TO THE SYBR ® SELECT MASTER MIX USER GUIDE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
© 2013 Life Technologies Corporation. All rights rerved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliates or their respective owners. AmpliTaq is a registered trademark of Roche Molecular Systems, Inc.
