Getting Started Guide Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Standard Curve Experiments Getting Started Guide Get Started Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Standard Curve Experiments Design the Experiment Prepare the Reactions Run the Experiment Analyze the Experiment © Copyright 2008, 2010 Applied Biosystems. All rights reserved. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS 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. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: Label License The StepOne™ and StepOnePlus™ Real-Time PCR Systems are covered by US patents and corresponding claims in their non-US counterparts, owned by Applied Biosystems Inc. No right is conveyed expressly, by implication, or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5′ nuclease methods. 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. TRADEMARKS: Applied Biosystems, AB (Design), MicroAmp, Primer Express, and VIC are registered trademarks, and FAM, JOE, ROX, StepOne, StepOnePlus, TAMRA, and VeriFlex are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. Macintosh is a registered trademark of Apple Computer, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Part Number 4376784 Rev. F 08/2010 Contents Preface . vii How to Use This Guide . vii How to Obtain More Information . ix How to Obtain Support . xii Safety Conventions Used in This Document . xiii Symbols on Instruments . xiv Safety Labels on Instruments . xvi General Instrument Safety . xvii Chemical Safety . xviii Chemical Waste Safety . xix Electrical Safety . xx LED Safety . xxi Biological Hazard Safety . xxi Workstation Safety . xxii Safety and Electromagnetic Compatibility (EMC) Standards . xxii Chapter 1 Get Started. 1 About the StepOne™ and StepOnePlus™ Systems . 2 Supported Consumables . 4 About Standard Curve Experiments . 6 How to Use This Guide . 9 About the Example Experiment . 10 Example Experiment Workflow . 12 Chapter 2 Design the Experiment . 15 Chapter Overview . 16 Create a New Experiment . 17 Define the Experiment Properties . 20 Define the Methods and Materials . 22 Set Up the Targets . 25 Set Up the Standards . 27 Set Up the Samples . 29 Set Up the Run Method . 31 Review the Reaction Setup . 33 Order Materials for the Experiment . 38 Finish the Design Wizard . 42 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Standard iii Curve Experiments Chapter 3 Prepare the Reactions . 45 Chapter Overview . 46 Prepare the Sample Dilutions . 47 Prepare the Standard Dilution Series . 49 Prepare the Reaction Mix . 51 Prepare the Reaction Plate . 53 Chapter 4 Run the Experiment . 59 Chapter Overview . 60 Prepare for the Run . 61 (Optional) Enable the Notification Settings . 63 Start the Run . 65 Monitor the Run . 69 Unload the Instrument and Transfer the Data . 76 Chapter 5 Analyze the Experiment . 81 Chapter Overview . 82 Section 5.1: Review Results . 83 Analyze the Experiment . 84 View the Standard Curve . 90 View the Amplification Plot . 92 View the Well Table . 99 Publish the Data . 102 Section 5.2: Troubleshoot (If Needed) . 103 View the Analysis Settings . 104 View the QC Summary . 106 Omit Wells from the Analysis . 108 View the Multicomponent Plot . 109 View the Raw Data Plot . 112 Appendix A Alternate Experiment Workflows . 115 Advanced Setup Workflow . 116 QuickStart Workflow . 117 Template Workflow . 119 Export/Import Workflow . 121 iv Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Standard Curve Experiments Bibliography . 125 Glossary . ..