Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems

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1 USER BULLETIN Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems Publication Number MAN Revision A This user bulletin is for use with the StepOne and StepOnePlus Real-Time PCR Systems. For detailed instructions, refer to the StepOne and StepOnePlus Real-Time PCR Systems Installation, Networking, and Maintenance Guide (Pub. no G). Custom dyes overview The StepOne and StepOnePlus Real-Time PCR Systems support the use of custom dyes (not supplied by Thermo Fisher Scientific) that excite at 470 nm and emit within the spectral range of nm. Do not use additional red dye with an emission between 623 ± 14 nm in combination with a master mix containing ROX. Custom dye calibration For each custom dye, determine the optimal dye concentration. Use this concentration for preparing all subsequent custom dye calibration plates. Prepare a custom dye dilution plate. q Run the dilution plate as an experiment. q Determine the optimal dye concentration. Custom dye dilution guidelines Prepare a 2 or 3 fold dilution series for each custom dye. Dilute the dye concentrations to a range of nm. Dispense µl per well for 96 well plates or 5 µl per well for 384 well plates. Dilute the dye in nuclease free water or in a buffer that is compatible with your master mix. (Intercalating dyes only) Add the appropriate amount of amplified PCR product to generate fluorescence. For Research Use Only. Not for use in diagnostic procedures.

2 Prepare a custom dye dilution plate Prepare a custom dye dilution plate IMPORTANT! Wear powder-free gloves throughout the procedure. 1. Prepare a 2 or 3 fold dilution series of the custom dye. 2. Dispense aliquots of each dilution into the center of a reaction plate, then seal the plate. A full plate is not needed. See the figures below for suggested replicates. 100 nm 200 nm 400 nm 800 nm 1600 nm 100 nm 200 nm 400 nm 800 nm 1600 nm 100 nm 200 nm 400 nm 800 nm 1600 nm 3. Vortex the plate for 5 seconds, then centrifuge for 2 minutes at 750 to 1000 g. 2 Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems

3 Prepare a custom dye dilution plate 4. Confirm that the liquid in each well is at the bottom of the well and free of bubbles. If it is not, centrifuge the plate again. GR well 2 GR well 1 GR well 3 IMPORTANT! Keep the bottom of the plate clean. Fluids and other contaminants on the bottom of the plate can contaminate the sample block and cause an abnormally high background signal. Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems 3

4 Run the dilution plate as an experiment Run the dilution plate as an experiment 1. Load the plate into the instrument. 2. Access Advanced Setup from the home screen of the desktop software in one of two ways. Click Advanced Setup. Note: If this option is not visible, click the down arrow under Design Wizard to expand the Set Up options. In the toolbar across the top, use the New Experiment drop-down list to select Advanced Setup. 3. Enter the experiment properties. a. In the Setup panel, click Experiment Properties. b. Enter an experiment name. c. Select the instrument you will use. 4 Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems

Determine the optimal dye concentration d. Select the Genotyping experiment type. e. Select Other as the reagents used to detect the target sequence. f. Select the Standard ramp speed. g.

5 Determine the optimal dye concentration d. Select the Genotyping experiment type. e. Select Other as the reagents used to detect the target sequence. f. Select the Standard ramp speed. g. Scroll to the bottom of the page and de-select inclusion of Pre-PCR Read and Amplification. Only a Post-PCR Read is needed. 4. Configure the plate setup. a. In the Setup panel, click Plate Setup. b. In the Assign Sample to the Selected Wells table, enter the dye concentration as the sample name. c. Assign a placeholder SNP assay to each well containing a sample. d. Select None as the passive reference. 5. Configure the method. a. In the Setup panel, click Run Method. b. Set the Post-PCR Read hold to 60 C for 2 minutes. c. Enter the appropriate reaction volume. 6. Click Save. 7. Click Start Run. 8. Download the experiment results. 9. Unload the plate from the instrument. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the plate temperature can reach 100 C. Allow it to cool to room temperature before handling. Determine the optimal dye concentration When the run is complete, review the dye signal data. 1. In the Analysis tab of the software, select Raw Data Plot. This plot displays the raw fluorescence signal of each dye, for individual wells over the duration of the PCR run. 2. For each replicate population of dilutions, select the wells in the View Plate Layout tab to view in the plot. Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems 5

6 Limited product warranty 3. Identify the well(s) yielding signals in the acceptable range of 250,000 1,000, Select the lowest concentration that falls within the acceptable signal range as the optimal dye concentration. Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at terms-and-conditions.html. If you have any questions, please contact Life Technologies at 6 Custom dye calibration for StepOne and StepOnePlus Real-Time PCR Systems

7 The information in this guide is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Thermo Fisher Scientific Inc. All rights reserved. For support visit thermofisher.com/support or thermofisher.com 7 October 2015

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