Agilent Brilliant III Ultra-Fast SYBR QPCR Master Mix for ABI 7500 Manual
Contents
1. to Purchaser Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser s own internal research No other patent rights are conveyed expressly by implication or by estoppel 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 SYBR is licensed for research and development only under patents and patent applications owned by Invitrogen Corporation SYBR is a registered trademark of Molecular Probes Inc Product Information Ordering Information Technical Services Catalog 600805 400 reactions By phone US only 800 424 5444 x3 By phone US only 800 894 1304 x2 Catalog 600816 4000 reactions On the web www genomics agilent com By email techservices agilent com For other countries please contact your local sales representative at www agilent com chem contactus Manual Part Number 5990 3068 Revision A Agilent Technologies Inc 2010 Printed May 1 2010 5990 5810EN 2 Brilliant IIl Ultra Fast SYBR Green QPCR Master Mix2. Stratagene Products Brilliant II Ultra Fast SYBR Green OPCR Master Mix Quick Reference Guide for the ABI 7500 Fast Real Time PCR System This quick reference guide provides an optimized protocol for using the Stratagene Brilliant IIT Ultra Fast SYBR Green QPCR Master Mix with the 7500 Fast Real Time PCR System from Applied Biosystems For detailed instructions refer to the full product manual Dilute the reference dye 1 500 using nuclease free PCR grade water Prepare the experimental reactions by combining the components of the reagent mixture in the order listed in the table below Prepare a single reagent mixture for replicate reactions plus at least one reaction volume excess using multiples of each component Reagent Mixture Nuclease free PCR grade water to bring final volume to 20 ul including DNA 10 ul of 2x SYBR Green QPCR Master Mix x pl of upstream primer at optimized concentration 200 500 nM x pl of downstream primer at optimized concentration 200 500 nM 0 3 ul of diluted reference dye Gently mix the reagent mixture without creating bubbles then distribute the mixture to the experimental reaction tubes Add x ul of experimental DNA to each reaction to bring the final reaction volume to 20 ul The table below lists a suggested quantity range for different DNA templates DNA Quantity per reaction Genomic DNA 5 pg 50 ng cDNA 0 5 pg 100 ng Refers to RNA input amo
3. unt during cDNA synthesis Mix the reactions without creating bubbles then centrifuge briefly phe Agilent Technologies Set Upthe 1 From the Home screen of the 7500 software click Advanced Setup QPCR Plate and 2 Complete the Setup screens for a new experiment as needed Thermal Profile On the Experiment Properties screen select SYBR Green Reagents including a melt curve and the Fast ramp speed 3 On the Run Method screen set the reaction volume to 20 ul and mark the Expert Mode check box Click Select View Filters and deselect any filters not in use in the experiment 4 Adjust the thermal profile according to the image below Reaction Volume Per Well 20 uL Y ExpertMode SelectfView Fitters Add Stage Add Step Delete Selected Undo Set Hold Time othina to Redo i Collect Data Y il Open Run Method Save Run Method Revertto Defaults Holding Stage Cycling Stage Melt Curve Stage Number ofCycles 40_ Continuous StepAndHold Enable AutoDelta Starting Cycle 95 0 C Step 4 Note If you do not require a high resolution melt curve you can select the StepAndHold option for the melt curve stage and then increase the ramp rate to 0 5 C per second to shorten the protocol time Run the PCR 1 Place the reactions in the 7500 instrument Program 9 Click START RUN Analyze Data 1 Analyze the results of the run as needed for your experiment Notices
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Agilent Brilliant III Ultra Fast SYBR QPCR Master Mix for ABI 7500 Manual