Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

Transcription

1 Quick Reference Card Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol Introduction Running the Axiom 2.0 Assay requires the following sets of steps: 1. Genomic DNA Prep, described in either the Axiom TM 2.0 gdna Sample Prep QRC (P/N ), the Axiom TM gdna Sample Prep for Genome-Wide BOS 1 Array Plate QRC (P/N ), or Chapter 2 in Section 1 Axiom TM Microbiome Assay Protocol in the Axiom TM Microbiome Solution User Guide (P/N ). 2. Automated Target Prep of the samples, performed using the NIMBUS TM. 3. Array Processing, described in GeneTitan TM MC Protocol for Axiom TM 2.0 Array Plate Processing QRC (P/N ). This QRC describes the automated target prep, performed using the NIMBUS TM. IMPORTANT: This QRC contains an abbreviated set of instructions. You must carefully read all the instructions in Chapter 3 Target Preparation with NIMBUS TM in the Axiom TM 2.0 Assay on the NIMBUS TM Target Preparation Instrument User Guide (P/N ) before running the Automated Target Prep Method. The Axiom TM 2.0 Assay on the NIMBUS TM Target Preparation Instrument User Guide covers the assay steps in more detail and provides information on running multiple plates per week through the automated target prep process. Stage 1 DNA Amplification Genomic DNA Plate Preparation We recommend that you prepare your genomic DNA sample plate in a clean room. The clean room should be separate from the laboratory where the Axiom 2.0 Assay is performed and should be free of DNA amplified in other procedures. 1. Performing DNA Amplification 1. Set the incubator/oven temperature at 37 C. 2. Set the centrifuge temp at room temperature. 3. Prepare reagents from Module 1 (P/N ) of the Axiom 2.0 Reagent Kit, as shown in Table 1: Table 1. Reagents Preparation for Stage 1 Reagent Temp Out of Module* Treatment Axiom 2.0 Amp Soln Thaw at Room Temp (~1 hr) Vortex for 30 sec to thoroughly mix Axiom Water Thaw at Room Temp Vortex Axiom 2.0 Denat Soln 10X Thaw at Room Temp Vortex and spin Axiom 2.0 Neutral Soln Thaw at Room Temp Vortex for 30 sec to thoroughly mix Axiom 2.0 Amp Enzyme Keep at 20 C Just before use, flick tube 3X, spin, and place in the cold block *Temp Out of Module: temperature reagent is held at immediately after removal from module 4. Thaw Sample Plate: NOTE: All human Axiom arrays (except the Axiom TM Genome-Wide Pan-African Array Set) require a total of 100 ng. The Axiom Genome-Wide Pan- African Array Set requires a total of 300 ng, or 100 ng per array (there are three arrays in the Axiom Genome-Wide Pan-African Array Set). Diploid plants and animals require 150 ng per array and polyploid plants and animals require 200 ng per array. For Axiom TM Microbiome, a total of 50 ng of gdna or 17.5 µl of cdna is required per array. 1. Bring your DNA samples to room temperature on the bench top. Pulse-spin plate to get all droplets down. 2. Leave at room temperature. 5. Run the NIMBUS method: 1. Select the DNA Amplification step, then click Run. For Research Use Only. Not for use in diagnostic procedures.

2 2. Set up the deck as indicated in the deck setup prompt and following screens, then click Run. NOTE: The deck setup is also shown in Figure 1 on the next page of this QRC. 6. When finished, remove the sample plate from the deck. 7. Blot the top of the plate with a Kimwipe. Tightly seal the plate. 8. Vortex and spin. 9. Place the sample plate in the preheated 37 C oven and incubate for 22 to 24 hr. 2. What to do Next After the incubation period, do one of the following: Proceed directly to Stage 2 Fragmentation. Tightly seal and store the sample plate at 20 C Figure 1. Stage 1 Deck Layout 2 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

3 Stage 2 Fragmentation Reagents and Samples Required Reagents from Module 2-1 (P/N ) and Module 2-2 (P/N ) of the Axiom TM 2.0 Reagent Kit; Isopropanol is user-supplied. Prepare as shown in Table 2 and place in the appropriate place on the deck. Table 2. Reagent Preparation for Stage 2 Fragmentation Reagent Temp Out of Treatment Module* Axiom 10X Frag Buffer Thaw at Room Temp Vortex Axiom Frag Diluent Place on ice Vortex and spin Axiom Frag Enzyme Keep at 20 C Just before use, flick tube 3X, spin, and place in the cold block Axiom Frag Rxn Stop Room Temp Vortex Axiom Precip Soln 1 Place on ice Vortex Axiom Precip Soln 2 Thaw at Room Temp Vortex and spin Isopropanol Not Applicable Room Temp Notes: *Temp Out of Module: temperature reagent is held at immediately after removal from module 1. If frozen, thaw the Sample Plate 1. Place the deep well plate in a small bath of room temperature Millipore water for ~50 min (until all wells have thawed). 2. Spin the plate down at 1000 rpm for 30 sec. 3. Remove the seal and blot the top of the plate with a Kimwipe. 4. Tightly reseal the plate with a fresh seal. 5. Vortex the plate for 30 sec to thoroughly mix. 6. Spin the plate down at 1000 rpm for 30 sec. 2. Fragment Samples 1. Run the NIMBUS method: 1. Select the Fragmentation step, then click Run. 2. Setup the deck as indicated the deck setup prompt and following screens, then click Run. NOTE: The deck layout is also shown in Figure 2 on the next page of this QRC. 3. Precipitate Samples 1. When the NIMBUS method is finished, remove the sample plate (Precipitation Plate; deck position 8) from the deck. 2. Blot the top of the plate with a Kimwipe. 3. Tightly seal the plate. 4. Place the plate in a 20 C freezer overnight to precipitate the DNA. 4. What to do Next After overnight precipitation proceed directly to Stage 3 Centrifugation and Drying Pellets. Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 3

4 Figure 2. Stage 2 Deck Layout 4 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

5 Stage 3 Centrifugation and Drying Pellets 1. Centrifuge and Dry Pellets 1. Preheat the oven to 37 C; cool centrifuge to 4 C. 2. Centrifuge the Precipitation Plate at 3200 xg (or rcf) at 4 C for 40 min. 3. Remove the seal, then invert the plate over a waste container to allow the liquid to drain. 4. While still inverted, gently press the top of the plate on a stack of Kimwipes. 5. Transfer and blot the plate onto a new pile of Kimwipes twice during the 5 min period. 6. Turn the plate right side up and place in the preheated oven for 20 min. 2. What to do Next After the pellets have been dried, do one of the following: 1. Proceed directly to Stage 4A Resuspension (even if some droplets of liquid remain). 2. If storing the plate for resuspension later in the same day: Tightly seal the sample plate. If resuspension will be carried out within 4 hours, keep the plates at room temperature. If resuspension will be carried out in more than 4 hours, store the plates in a refrigerator (2-8 C). 3. If storing the plate for resuspension on another day. Tightly seal the sample plate and store at 20 C. Stage 4A and 4B Resuspension and Hybridization Preparation NOTE: The Resuspension step should be immediately followed by Hybridization Preparation. (See Stage 4B Hybridization Preparation). We recommend thawing the Module 2-1 and Module 2-2 reagents before starting Stage 4A to minimize any time lapses between Stage 4A Resuspension and Stage 4B Hybridization Preparation. Reagents Required for Both Resuspension and Hybridization Preparation Prepare Reagents from Module 2-1 (P/N ) and Module 2-2 (P/N ) of the Axiom TM 2.0 Reagent Kit. Table 3. Reagent Preparation for Stage 4A Resuspension and 4B Hybridization Preparation Reagent Temp Out of Module* Treatment Stage Axiom Hyb Buffer Place on ice Vortex 4B Axiom Hyb Soln 1 Thaw at Room Temp Vortex and spin 4B Axiom Resusp Buffer Warm to Room Temp (~1 hr) Vortex 4A Axiom Hyb Soln 2 Place on ice Vortex and spin 4B *Temp Out of Module: temperature reagent is held at immediately after removal from module Stage 4A Resuspension NOTE: The Resuspension step should be immediately followed by Hybridization Preparation. 1. Prepare the Precipitation Plates Before Proceeding with Resuspension: Plates stored at 2-8 C after Stage 3 should be allowed to warm to room temperature for 30 min. Plates frozen at 20 C after Stage 3 should be allowed to equilibrate at room temperature for 1.5 hr. 2. Add Resuspension Buffer to DNA Pellets 1. Run the NIMBUS method: 1. Select the Resuspension step, then click Run. 2. Setup the deck as indicated in the NIMBUS deck setup prompt and following screens, then click Run. NOTE: The deck setup is also shown in Figure 3 on the next page. Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 5

6 3. Resuspend by Off-Deck Shaking 1. When the NIMBUS method is finished, remove the sample plate (Resuspension Plate; deck position 5) from the deck. 2. Blot the top of the plate with a Kimwipe. 3. Seal the plate tightly; blue pellets should be visible at the bottom of the wells. 4. Place the sample plate onto one of the following shakers for 10 minutes: Thermo Scientific Compact Digital Microplate Shaker: set at 900 rpm Jitterbug: set at speed of 7 5. Inspect the plate from the bottom. If the pellets are not dissolved, repeat the shaking step. 6. Pulse-spin the plate in a room temperature centrifuge. 4. What to do Next Proceed to Stage 4B Hybridization Preparation. Figure 3. Stage 4A Deck Layout 6 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

7 Stage 4B Hybridization Preparation IMPORTANT: Be sure to perform the off-deck shaking to resuspend the samples before continuing on to this stage. 1. Hybridization Preparation 1. Run the NIMBUS method: 1. Select the Hybridization Preparation step, then click Run. 2. Setup the deck as indicated in the NIMBUS deck setup prompt and following screens, then click Run. NOTE: The deck setup is also shown in Figure 4 on the next page. 2. What to do Next Do one of the following: Proceed directly to Stage 4C Sample QC. Tightly seal the Hyb Ready sample plate and store at 20 C. Figure 4. Stage 4B Deck Layout Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 7

8 Stage 4C Sample QC Reagents Required Invitrogen TrackIt 25 bp DNA Ladder and Cyan/Orange Gel Loading Buffer reagents are user-supplied. Table 4. Reagent Preparation for Stage 4C Sample QC Reagent Nuclease-free water Gel Diluent: Invitrogen TrackIt Cyan/Orange Gel Loading Buffer (diluted 1:1000) Invitrogen TrackIt 25 bp DNA Ladder (diluted 1:15) 1. Prepare the Hyb Ready Plate If the Sample Plate is Frozen Treatment Not Applicable See Gel QC Instructions See Gel QC Instructions Thaw and Prepare the Hyb Ready Plate 1. Remove Hyb Ready plate from 20 C storage. 2. Ensure the plate is tightly sealed. 3. Vortex the plate for 30 seconds to thoroughly mix. 4. Pulse-spin plate to 1000 rpm. If Proceeding Directly from Stage 4B Prepare the Hyb Ready Plate 1. Confirm the plate is tightly sealed. 2. Vortex the plate for 1 sec each corner, and 1 sec in the center at the maximum setting. 3. Pulse-spin plate to 1000 rpm. 2. Prepare Gel Diluent The TrackIt Gel Loading Buffer, diluted 1000-fold, is referred to Gel Diluent in this method. To Dilute the TrackIt Cyan/Orange Loading Buffer: 1. Add 50 µl of TrackIt Cyan/Orange Loading Buffer to ml nuclease-free water. Total volume 50 ml. 2. Mix well. 3. Store at room temperature. 3. Run the NIMBUS Method 1. Select the Sample QC step, then click Run. 2. Setup the deck as indicated in the NIMBUS deck setup prompt and following screens, then click Run. NOTE: The deck setup is also shown in Figure 6 on the next page. 4. Run Fragmentation QC Gels 1. Tightly seal the Gel QC plate, vortex, and spin. 2. Onto a 4% agarose e-gel, load: 20 µl from each well of the Gel QC plate. 15 µl diluted TrackIt 25 bp ladder to marker wells. 20 µl water to any unused wells. NOTE: Running 48 samples (one E-Gel) per plate is recommended. 3. Run for 22 min. 4. Review gel image (see Figure 5). 8 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

9 Figure 5. Gel Image 5. Quantitate the Resuspended Samples 1. Quantitate the samples prepared in the OD plate. 2. Assess the OD reading for each sample. Median Yield = 1200 µg/well 6. What to do Next Do one of the following: If the GeneTitan MultiChannel instrument is available, and if the gel QC and quantitation results were acceptable, proceed to Stage 5 Prepare Hybridization Tray. Tightly seal the Hyb Ready plate and store at 20 C. Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 9

10 Figure 6. Stage 4C Deck Layout 10 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

11 Stage 5 Prepare Hybridization Tray Reagents and Samples Required Reagents from Module 3 of the Axiom TM 2.0 Reagent Kit. Table 5. Reagent Preparation for Stage 5 Prepare Hybridization Tray Reagent Temp Out of Module* Treatment Axiom Wash Buffer A (both bottles; 1L) Room Temperature Invert 2-3X for mixing before filling GT bottle Axiom Wash Buffer B Room Temperature Invert 2-3X for mixing before filling GT bottle Axiom Water Room Temperature N/A *Temp Out of Module: temperature reagent is held at immediately after removal from module Materials and Instruments Required Wash buffers from the Axiom 2.0 Reagent Kit Axiom Wash A, P/N Axiom Wash B, P/N Axiom Water, P/N Hyb Ready plate from Stage 4B, Hybridization Preparation Axiom 96 array plate or Axiom TM Microbiome 96 Array Plate Hybridization Tray (P/N ) from the Axiom TM Array GeneTitan TM Consumables Kit (P/N ) GeneTitan TM MC Instrument available for hybridization NIMBUS TM Thermal cycler (See Axiom TM 2.0 Assay for NIMBUS TM Site Preparation Guide, P/N , for recommended models) Program with the Axiom 2.0 Denature protocol of: 95 C for 10 min; 48 C for 3 min; hold at 48 C. Use the heated lid option when setting up or running protocols. 1. Prepare the Hyb Ready Sample Plate (if Stored at 20 C) and the Array Plate To Prepare the Sample Plate: 1. Warm up the Hyb Ready plate at room temperature for 5 minutes. 2. Make sure the Hyb Ready plate is sealed well. If the plate is not sealed well: a. Spin the plate and carefully remove the old seal. b. If there is condensation on the top of the plate, blot dry gently with a Kimwipe. c. Use a fresh seal and tightly reseal the plate. 3. Vortex the Hyb Ready plate briefly, then pulse-spin to 1000 rpm. 4. Place the Hyb Ready plate at room temperature. To Prepare the Array Plate: 1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument. a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature. b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file using AGCCPortal (see Stage 1 Create and Upload Batch Registration File in Chapter 4 of the Axiom TM 2.0 Assay Automated Workflow on NIMBUS TM Method version User Guide, P/N ). 2. Prepare the GeneTitan TM MC Instrument 1. Prepare the reagents from Module 3 by inverting bottles 2-3X to mix (Axiom Wash Buffer A, Axiom Wash Buffer B, and Axiom Water). 2. Launch AGCC and set up the GeneTitan MC Instrument to run the Hyb-Wash-Scan Setup option. For more information, see: GeneTitan TM MC Protocol for Axiom TM 2.0 Array Plate Processing QRC, P/N Chapter 4, Array Processing with the GeneTitan TM Multi-Channel Instrument in the Axiom TM 2.0 Assay on the NIMBUS TM Target Preparation Instrument User Guide, P/N Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 11

12 3. Perform Sample Denaturation 1. Place Hyb Ready plate in thermal cycler block, secure lid and start the Axiom 2.0 Denature program. 2. Once the Axiom 2.0 Denature program reaches completion, promptly remove the plate from the cycler and place the denatured samples on deck position Run the NIMBUS Method 1. During sample denaturation, run the Prepare Hybridization Tray step, then click Run. 2. Setup the tips and hyb tray on the deck as indicated in the Place Labware deck setup prompt. Promptly continue with the deck setup immediately after the denatured hyb ready sample plate is placed on the deck. NOTE: The deck setup is also shown in Figure 7 on the next page. 3. When the method is complete, remove the hyb tray from the deck. 4. Examine the hyb tray to ensure that there are no air bubbles present. Puncture any air bubbles that you may see using a clean pipette tip. 5. Load the array plate and hyb tray into the GeneTitan MC Instrument.Hybridization will continue on the GeneTitan MC Instrument for 23.5 to 24 hours. 5. What to do Next When the hybridization step on the GeneTitan MC Instrument is approximately 1.5 hours from completion (22 hours after the start of hybridization), proceed to Stage 6 Prepare GeneTitan TM Reagent Plates. Figure 7. Stage 5 Deck Layout 12 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

13 Stage 6 Prepare GeneTitan TM Reagent Plates IMPORTANT: The reagent trays prepared are for use with an Axiom array plate that is already in the GeneTitan MC instrument and is completing the hybridization stage. The method for Stage 6 Prepare GeneTitan Reagent Plates consists of two parts: Part 1: Preparation of the Scan Tray, Stain 2 Tray, and the Stabilize Tray. Part 2: Preparation of the Stain 1-1 and Stain 1-2 Trays and the Ligation Tray. After Part 1 of the method is completed, a dialog window for Labware Change (see Figure of the user guide) appears, prompting the user to remove labware from specific deck positions and replace them with new labware. Once finished with the labware change, the run will proceed with Part 2 of the method. 1. Prepare the reagents from Module 4 of the Axiom 2.0 Reagent Kit, as shown in Table 6 below: Table 6. Reagent Preparation Reagent Temp Out of Module* Treatment Module 4-1, 20 C (P/N ) Axiom Ligate Buffer Thaw at Room Temp 1. Place on bench top at room temp for 30 min or in a bath with room temperature Millipore water. 2. Vortex twice 3. Examine for precipitate. If any, warm bottle with your hands and vortex again for thirty seconds Axiom Ligate Enzyme Keep at 20 C until ready to use Just before use: 1. Flick 2 to 3 times to mix 2. Pulse-spin 3. Place in the cold block Axiom Ligate Soln 1 Thaw at Room Temp Vortex and spin Axiom Probe Mix 1 Thaw at Room Temp Vortex and spin Axiom Stain Buffer Thaw at Room Temp Vortex and spin Axiom Stabilize Soln Thaw at Room Temp Vortex and spin Module 4-2, 2-8 C (P/N ) Axiom Ligate Soln 2 Thaw at Room Temp (do not Vortex and spin place on ice) Axiom Probe Mix 2# Place on ice Flick 2 to 3 times to mix, then spin Axiom Wash A Leave on bench 1. Vortex twice 2. Place on Bench for 30 min 3. Look for precipitate 4. Vortex again if necessary Axiom Stain 1-A# Place on ice Flick 2 to 3 times to mix, then spin Axiom Stain 1-B# Place on ice Flick 2 to 3 times to mix, then spin Axiom Stain 2-A# Place on ice Flick 2 to 3 times to mix, then spin Axiom Stain 2-B# Place on ice Flick 2 to 3 times to mix, then spin Axiom Stabilize Diluent Place on ice 1. Vortex and spin 2. Look for precipitate. If any, warm tube to room temperature and vortex again Axiom Water Leave on bench N/A Axiom Hold Buffer# Room Temp Vortex Notes: * Temp Out of Module: temperature the reagent is held at immediately after removal from module. # These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time. N/A: not applicable in this case 2. Run the NIMBUS method: 1. Select the Prepare GeneTitan Reagent Plates step, and click Run. 2. Setup the deck as indicated in the deck setup prompt and following screens, then click Run. The deck setup for Part 1 is also shown in Figure 8 of this QRC. IMPORTANT: Label the stain trays and treat them with the antistatic gun. Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 13

14 3. Midway through the method, a second deck layout screen appears. Complete the labware change on the NIMBUS deck, as illustrated in Figure 9 of this QRC. 3. Prepare the GeneTitan TM Multi-Channel Instrument. See GeneTitan TM MC Protocol for Axiom TM 2.0 Array Plate Processing QRC (P/N ). 4. Treat the stain and scan tray lids with the antistatic gun. 5. When the method is complete, examine each tray to: Ensure that all wells contain reagents (manually add if not present) Puncture any bubbles with a clean pipette tip. 6. Cover the reagent trays and scan tray with lids. 7. Immediately load the reagent and scan trays into the the GeneTitan MC Instrument. See GeneTitan TM MC Protocol for Axiom TM 2.0 Array Plate Processing QRC (P/N ). IMPORTANT: Droplets close to or on the top of the well dividers may cause the lid to stick to the tray during GeneTitan processing. If necessary, blot-dry. 14 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

15 Figure 8. Stage 6 Initial Deck Layout Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol 15

16 Figure 9. Stage 6 Labware Change Deck Layout Stage 6 Prepare GeneTitan TM Reagent Plates Deck Layout, Part 2, Labware Change 1. Remove the following labware: Deck position 4: Scan Tray Deck position 5: Stain 2 Tray Deck position 8: Stabillize Tray 2. Discard the following labware: Deck position 7: 4-Column Reservoir and Frame 3. Add labware and reagent as indicated below. 16 Axiom TM 2.0 for NIMBUS TM Automated Target Prep Protocol

17 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 Product documentation, including: - User guides, manuals, and protocols - Certificates of Analysis - Safety Data Sheets (SDSs; also known as MSDSs) 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. 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