Eureka Genotyping Assay

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1 Quick Reference Card Eureka Genotyping Assay This quick reference card is intended for experienced users. For detailed instruction, please refer to the Eureka Genotyping Workflow User Guide (P/N Rev. 1). For a full list of reagents and materials required for the assay, please refer to the Eureka Genotyping Solution Site Preparation Guide (P/N Rev.1). All reagents need to be thawed at room temperature prior to use. INTEGRA VIAFLO 384 Instrument The Eureka Genotyping Assay utilizes the INTEGRA VIAFLO 384 pipetting instrument. Below is a diagram of the instrument identifying the various locations of instrument components. While conducting the Eureka Genotyping Assay it is important to place the various plates and cooling blocks in the correct plate holder positions. Please refer to the diagram below for clarification, if needed. Stage 1 DNA Heat Fragmentation Materials Required Twin.Tec TM PCR Plate 384 (green) Aluminum heat seal Prepare Genomic DNA Plate 1. Start with generally salt-free genomic DNA of at least 42 ng/μl in TE, ph Dispense 3.5 μl of gdna into a green 384 plate with four wells containing supplied control DNA. 3. Heat seal, spin down. 4. Use the EG_Heat_Frag thermal cycler program. 5. Leave the plate at 25 C for no longer than 30 min. For Research Use Only. Not for use in diagnostic procedures.

EG_Heat_Frag Thermal Cycler Program Temperature Time 98 C 15 min 25 C Hold Stage 2 Hybridization with Eureka Genotyping Panel Eureka Genotyping Panel (2.

2 EG_Heat_Frag Thermal Cycler Program Temperature Time 98 C 15 min 25 C Hold Stage 2 Hybridization with Eureka Genotyping Panel Eureka Genotyping Panel (2.5 ml) Green 384 PCR Plate containing Fragmented DNA 12 Column Reservoir Twin.Tec PCR Plate 384 (blue) 2 Pre-chilled VIAFLO PCR 384 Cooling Plates 12.5 μl filtered GripTips Aluminum heat seal 1. Invert the Eureka Ligation Solution multiple times to mix do not vortex. Pour the Eureka Genotyping Panel into the far right column of the reservoir. 2. On the VIAFLO: Position A: Place the 12 column reservoir with the liquid on the right side. Position B: Place a VIAFLO cooling plate with an empty blue 384 plate. 3. Run VIAFLO program EG_PROBEDISP. 4. On the VIAFLO: Position A: Place the unsealed blue 384 plate on the VIAFLO cooling plate. Position B: Place the spun down and unsealed green 384 plate on the VIAFLO cooling plate. 5. Run VIAFLO program EG_PROBETODNA. 6. Heat seal the green 384 plate. Spin down to eliminate bubbles at the bottom of the wells. 7. Place into the thermal cycler and start EG_Hyb program. EG_Hyb Thermal Cycler Program Temp Time Cycles 98 C 1 min 60 C 20hrs. May require hold step or multiple cycles to achieve 20 hrs total) 54 C 1 min 2 Eureka Genotyping Assay

3 Stage 3 Ligation Eureka Ligation Solution (5.5 ml) 12 Column Reservoir Twin.Tec PCR Plate 384 (yellow) 2 Pre-chilled VIAFLO PCR 384 Cooling Plates Green 384 PCR Plate containing Fragmented DNA and Genotyping Panel 12.5 μl filtered GripTips Aluminum heat seal 1. Invert the Eureka Ligation Solution multiple times to mix do not vortex. Pour into the far right column of a 12 column reservoir. 2. On the VIAFLO: Position A: Place the 12 column reservoir with ligation solution. Position B: Place an empty yellow 384 PCR plate on the VIAFLO cooling plate. 3. Run VIAFLO program: EG_LIGDISP. 4. The yellow 384 plate can be kept on ice for up to 15 min before proceeding. 5. When EG_Hyb program completes on the hybridized green 384 plate, place the plate immediately on a pre-chilled VIAFLO cooling plate. Allow plate to chill for 1 min. 6. On the VIAFLO: Position A: Place the green hybridization plate on a chilled VIAFLO cooling plate. Position B: Place the yellow plate on a chilled VIAFLO cooling plate. 7. Run VIAFLO program EG_HYBTOLIG. 8. Heat seal, vortex and place back on cooling plate for 1 min. Spin down the yellow 384 plate. 9. Prestart thermal cycler program EG_Lig. When thermal cycler reaches 54 C, place the yellow ligation plate into the thermal cycler and continue the program. EG_Lig Thermal Cycler Program Temperature Time 54 C 15 min 92 C 15 sec 8 C Hold 10. Proceed to Stage 4: PCR Amplification. OPTIONAL STOPPING POINT: The post-ligation plate can be stored at 4 C overnight or at 20 C for up to one week. Eureka Genotyping Assay 3

Stage 4 PCR Amplification Eureka Sample Index Plate Eureka PCR Master Mix (3.5 ml) 12 Column Reservoir (from Stage 3) 2 Pre-chilled VIAFLO PCR 384 Cooling Plates Twin.

4 Stage 4 PCR Amplification Eureka Sample Index Plate Eureka PCR Master Mix (3.5 ml) 12 Column Reservoir (from Stage 3) 2 Pre-chilled VIAFLO PCR 384 Cooling Plates Twin.Tec 384 PCR plate (red) that becomes the Red 384 PCR Plate containing Sample Index PCR Solution Yellow 384 PCR Plate containing Ligation Reaction 12.5 μl filtered GripTips Aluminum heat seal 1. Take the thawed Eureka Sample Index Plate and spin down. 2. Enter Genotyping Panel and Sample Index Plate barcode into the Eureka Analysis Suite software. 3. Invert the thawed Eureka PCR Master Mix several times to mix. Pour the contents into the far right column of the rotated 12 column reservoir. 4. On the VIAFLO: Position A: Place the 12 column reservoir. Position B: Place an empty red 384 plate on the VIAFLO cooling plate. 5. Run VIALFO program EG_PCRMIXDISP. 6. Leave the red 384 plate in Position B. Discard the 12 column reservoir from Position A. 7. Load a VIAFLO cooling plate and Eureka Sample Index Plate into Position A. 8. Run VIAFLO program: EG_SMPLEINDX. 9. On the VIAFLO: Position A: Place the yellow 384 ligation reaction plate on a chilled VIAFLO cooling plate. Position B: Put the prepared red sample index plate on a chilled VIAFLO cooling plate. 10. Run VIAFLO program EG_LIGTOPCR. 11. Heat seal the red 384 PCR plate and spin down to eliminate bubbles. 12. Place into the thermal cycler and start program EG_PCR. EG_PCR Thermal Cycler Program Temperature Time Cycles 94 C 5 min 1 94 C 10 sec 65 C 30 sec C 1 min 1 8 C Hold 1 OPTIONAL STOPPING POINT: The post-pcr plate can be stored at 4 C overnight or at 20 C for up to one week. 4 Eureka Genotyping Assay

Post PCR Library Pooling TE ph 8.0 DNA Binding Buffer DNA Wash Buffer 10 mm Tris HCl ph 8.5 384 Individual Well Reservoir Zymo-Spin V Column 50 ml conical tube 2 ml collection tubes 1.

5 Post PCR Library Pooling TE ph 8.0 DNA Binding Buffer DNA Wash Buffer 10 mm Tris HCl ph Individual Well Reservoir Zymo-Spin V Column 50 ml conical tube 2 ml collection tubes 1.5 ml LoBind microfuge tubes Red 384 PCR Plate containing Sample Index PCR Reaction Pooling the Library 1. Tape 384 Individual Well Reservoir on top of unsealed red 384 plate containing PCR reaction. 2. Invert plate combo and spin at 800 xg for 1 min. 3. Avoid spilling; do not tilt plate. Clean-up and Concentration 1. Label Zymo-Spin V column, tighten bottom portion to ensure firm attachment and place in 50 ml conical tube. 2. Add 10 ml of DNA Binding Buffer to the pooled library in reservoir, gently mix and transfer all contents to labeled Zymo-Spin V column placed in 50 ml tube. 3. Spin 90 sec at 2500 rpm (~500 xg) and discard flow through. Repeat spin to make sure all sample has flowed through the column and discard flow through. 4. Perform two wash steps with 5 ml of DNA Wash Buffer, spinning at 2500 rpm (~500 xg) for 90 sec and discard flow through after each step. 5. Transfer bottom portion of the Zymo-Spin V column to 2 ml collection tube. 6. Perform two wash steps with 600 μl of DNA Wash Buffer, spinning at rpm for 1 min and discard flow through after each step. 7. Dry spin at rpm for 5 min in a new empty 2 ml collection tube. 8. Transfer the column into a new 1.5 ml LoBind microfuge tube and elute the DNA by adding 150 µl of TE ph 8.0 (NOT the Zymo Elution Buffet) to the column. 9. Incubate for 1 min and spin rpm for 1 min. OPTIONAL STOPPING POINT: The pooled library can now be stored at 20 C for up to 2 weeks. Eureka Genotyping Assay 5

6 Library Qualification and Quantification 1. Dilute library stock with 5 μl of pooled library and 20 μl of 10 mm Tris-HCl ph Quantify on Agilent DNA1000 assay run 2 wells per diluted library stick. 3. Calculate nm concentration and enter value of the diluted library into Eureka Genotyping Calculator. 6 Eureka Genotyping Assay

7 Documentation and support Customer and technical support Visit thermofisher.com/support for the latest in services and support, including: Worldwide contact telephone numbers Product support, including: - Product FAQs - Software, patches, and updates Order and web support 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 If you have any questions, please contact Life Technologies at thermofisher.com/support. Product documentation, including: - User guides, manuals, and protocols - Certificates of Analysis - Safety Data Sheets (SDSs; also known as MSDSs) Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer. 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. Corporate entity: Life Technologies Carlsbad, CA USA Toll Free in USA Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. All other trademarks are properties of their respective owners. P/N For support visit thermofisher.com/support or techsupport@lifetech.com thermofisher.com 23 January 2017

Eureka Genotyping Solution

Eureka Genotyping Solution Site Preparation Guide For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. DISCLAIMER TO THE EXTENT