DRAFT. Apollo 324 System. Protocol. PrepX-32i DNA Library

Transcription

1 Apollo 324 System PrepX-32i DNA Library Protocol

2 Copyright 2012, IntegenX Inc. All rights reserved. Information in this document is subject to change without notice. IntegenX assumes no responsibility for any errors that may appear in this document. RESEARCH USE ONLY The Apollo 324 System is for research use only. WARRANTY For warranty and liability information, please see IntegenX terms and conditions of sale. TRADEMARKS Apollo 324, BeadX and PrepX are trademarks of IntegenX Inc. (IXI) AMPure and Agencourt are registered trademarks of Beckman Coulter, Inc. All other trademarks are the sole property of their respective owners. IntegenX Inc Stoneridge Drive, Suite 300 Pleasanton, CA Part Number P036873, Rev. A 4/2012

3 Contents Chapter 1 Overview About the Protocol Workflow Processing Schematic Chapter 2 Preparing Samples and Reagents Overview Materials for Operation Reagent Kit Instruments Customer-Supplied Reagents and Consumables Preparing the Library Kit Reagents Preparing 5X AMPure XP Beads Preparing Adapter Mix Preparing 70% v/v EtOH Preparing the Molecular Biology Grade Water Aliquot Preparing Samples Preparing Ligation Mix Chapter 3 Setting Up and Running the Protocol Overview Setting Up a Run Work Surface Layout Launching the Software Placing Consumables on the Work Surface Loading Samples and Reagents Running the Protocol Starting a Run Monitoring a Run Finishing a Protocol Retrieving and Handling the Processed Libraries PCR and Post-PCR Conditions Reagent and Reaction Locations Index PrepX-32i DNA Library Protocol iii

4 Contents iv PrepX-32i DNA Library Protocol

5 Chapter 1 Overview In this chapter: About the Protocol About the Protocol 1 Workflow 2 Processing Schematic 2 This PrepX-32i DNA Library Protocol provides the basic information necessary to prepare indexed DNA libraries from 32 samples on your Apollo 324 System. This document assumes that you know how to use the Apollo 324 System and the touchscreen interface. For details on using the system, refer to the Apollo 324 System User Guide. IMPORTANT: IntegenX recommends that first-time users take advantage of user training offered with the installation of the system. A training video is available at The PrepX-32i DNA Library protocol reagent kit provides researchers the flexibility to optimize the performance of the Apollo 324 System to prepare up to 32 indexed DNA libraries in one run. The entire process is completed in approximately 7.1 hours, with the products ready for sequencing or amplification. The reagent kit is available for the Illumina Genome Analyzer and HiSeq 2000 NGS systems. Reagent kits are tailored for the instruments in which the samples will be analyzed. The kit includes bead mix strips, enzymes and buffers for 96 samples. PrepX-32i DNA Library Protocol 1

6 Chapter 1 Overview Workflow Workflow 1. Press the PrepX-32i DNA Library button on the touchscreen to activate the Peltier heating/cooling units. 2. Place consumables, reagents, magnetic beads and samples in the racks on the work surface. 3. Start the protocol run, using the touchscreen interface. 4. The 32 samples undergo BeadX processing: a. End repair b. Intermediary bead-based cleanup c. A-Tail d. Intermediary bead-based cleanup e. Adapter ligation 5. Final double bead-based cleanup and size selection is performed. 6. DNA samples are now ready for amplification and can be placed into the Agilent Bioanalyzer or equivalent DNA analyzer to determine the concentration after being centrifuged briefly. Reaction Cleanup Fragmented DNA Samples End Repair Bead Cleanup End Repair Master Mix Elution A-Tail Bead Cleanup A-Tail Master Mix Elution Ligation High Cut Adapter Ligation Master Mix Flow Through Low Cut Size-Selected Libraries Indexed Processing Schematic 2 PrepX-32i DNA Library Protocol

7 Chapter 2 Preparing Samples and Reagents Overview In this chapter: Overview 3 Materials for Operation 3 Preparing the Library Kit Reagents 5 Preparing 5X AMPure XP Beads 6 Preparing Adapter Mix 6 Preparing 70% v/v EtOH 6 Preparing the Molecular Biology Grade Water Aliquot 6 Preparing Samples 7 Preparing Ligation Mix 7 This chapter describes how to prepare samples and reagents for the PrepX-32i DNA Library Kit protocol. IMPORTANT: We only guarantee the Apollo 324 System to perform using the recommended supplies and materials listed in this document. Materials for Operation Reagent Kit The following reagents are provided in the PrepX-32i DNA Library Reagent Kit. The kit provides sufficient reagents for three 32-sample runs (96 samples). BeadX Bead Mix 24 Bead Mix strips (4 tubes in each strip, black labeled seal) containing BeadX reagents and BeadX buffers Enzymes 24 Enzyme strips (four tubes in each strip, orange labeled seal) Ligase and Buffer Ligase enzyme (three tubes) Ligase buffer (three tubes) EDTA 0.5M, ph 8.0 Molecular biology grade water PrepX-32i DNA Library Protocol 3

8 Chapter 2 Preparing Samples and Reagents Materials for Operation Instruments The following items are required. Customer-Supplied Reagents and Consumables Item Part Number Supplier 96-well thermocycler various various Agilent 2100 Bioanalyzer G2938C Agilent Agilent High Sensitivity DNA Kit (to perform Agilent Bioanalyzer runs for your prepared samples) Centrifuge for 0.2 ml 8-tube strips various various Vortex mixer for preparing reagents and samples various various Heat sealer various various Heat sealer fixture (8-well cold block) IntegenX The following reagents and consumables are recommended to run the Apollo 324 System. Customer-Supplied Consumables Part Number Supplier 1.1 ml 12-tube strips (Axygen ) VWR 0.2 ml 8-tube strips (Axygen) VWR 0.2 ml 8-cap strips (Axygen) VWR Eppendorf 96-well skirted microtiter plate, conical, 150 µl VWR 50 ml Falcon test tube VWR 15 ml Falcon test tube VWR 2 ml screw cap test tube VWR 1.5 ml DNA LoBind tube Eppendorf Easy Pierce Heat Sealing Foil (aluminum, 20 microns) AB1720 Thermo Fisher Scientific Piercing tips IntegenX Dispensing filter tips IntegenX Reservoirs IntegenX Micropipette tips (20 µl, 200 µl and 1000 µl) various various Micropipettes (20 µl, 200 µl and 1000 µl) various various Customer-Supplied Reagents Part Number Supplier 100% EtOH, reagent grade E7023 Sigma-Aldrich AMPure Beads (450 ml kit) A63882 Agencourt Ligation Adapter Mix (1+2, annealed) (custom order) IDT 4 PrepX-32i DNA Library Protocol

9 Preparing the Library Kit Reagents Chapter 2 Preparing Samples and Reagents The recommended initial purchase quantities and usage per run of the consumables are as follows: Requirement Recommended Initial Purchase Usage per Run 1.1 ml 12-tube strips 1 box 1 rack per run 0.2 ml 8-tube strips 1 box 6 runs samples 0.2 ml 8-cap strips 1 box 6 runs samples Eppendorf 96-well skirted microtiter plate, conical, 150 µl 1 box 25 runs samples 50 ml Falcon test tube n/a varies 15 ml Falcon test tube n/a varies 2 ml screw cap test tubes n/a varies 1.5 ml DNA LoBind tube n/a varies Easy Pierce Heat Sealing Foil (aluminum, 20 microns) Preparing the Library Kit Reagents To prepare the reagents: 1 package varies Piercing tips 1 box of runs - 24 tips/run Dispensing filter tips 1 box of runs tips/run Reservoirs 1 box of runs - 4 reservoirs/run Micropipette tips (20 µl, 200 µl and 1000 µl) n/a varies Micropipettes (20 µl, 200 µl and 1000 µl) n/a varies Safety glasses, gloves and lab coats as required in your lab n/a varies 1. Retrieve the box containing the beads from the refrigerator. Retrieve the box containing the enzymes from the freezer and thaw the enzymes. Only thaw the enzymes that you will be using for the run. Keep all reagents, including ligase enzyme, ligase buffer and annealed adapters on ice. 2. Vortex and spin down reagents briefly. For IntegenX reagents, use an 8-strip adapter for the micro-centrifuge. 3. Visually inspect to be sure that the entire volume is at the bottom of each vial with no air pockets. PrepX-32i DNA Library Protocol 5

10 Chapter 2 Preparing Samples and Reagents Preparing 5X AMPure XP Beads Preparing 5X AMPure XP Beads Preparing the beads can be done in advance. 1. Mix the AMPure bead stock. Make sure that all of the settled beads are well homogenized. 2. Pipette 20 ml of 1X AMPure into a 50 ml test tube, magnetically pellet the beads and remove 16 ml of supernatant. 3. Vortex the remaining 4 ml of 5X bead solution to resuspend the beads. IMPORTANT: Just before starting the protocol, you will aliquot the AMPure beads into an empty 8-tube strip on the work surface. This step is done last in order to avoid bead settling. Preparing Adapter Mix NOTE: This section applies only if you are preparing your own adapter mix. 1. Resuspend lyophilized adapter oligonucleotides to 100µM each in 10mM Tris-EDTA, ph Add equal volumes of adapters to a 1.5 ml microfuge tube to produce a 50µM mixture of both, then vortex and spin down briefly. 3. Aliquot 50 µl of the adapters into PCR tubes, cap the tubes and anneal using the following thermocycling parameters: Step 1: 95 C for 2:00 minutes Step 2: 0.1 C/second to 25 C Step 3: 25 C for 5:00 minutes Step 4: 4 C hold 4. Pool the annealed adapters into a clean 1.5 ml tube. 5. Vortex and spin down briefly. 6. Aliquot into single use portions in PCR tubes and cap the tubes. Store frozen at -20 C. NOTE: Adapters can be prepared in advance and stored at -20 C prior to use, but avoid freeze-thaw cycles. 7. Prepare the use-concentration by diluting 10-fold (50µM to 5µM) in 10mM Tris or reagent grade water. Preparing 70% v/v EtOH Combine 28 ml of 100% EtOH and 12 ml of UltraPure water in a sterile 50 ml Falcon tube and mix. Preparing the Molecular Biology Grade Water Aliquot Pipette 25 ml of molecular biology grade water into a sterile 50 ml Falcon tube. 6 PrepX-32i DNA Library Protocol

11 Preparing Samples Chapter 2 Preparing Samples and Reagents Preparing Samples NOTE: We recommend using color-coded Axygen strip tubes to ensure correct setup. 1. Pipette 15 µl of fragmented DNA sample, 200 ng (13 ng/µl) to 1000 ng (67 ng/µl) concentration, into each tube of an Axygen 8-tube strip. 2. Repeat step 1 three times to yield four 8-tube strips filled with sample. 3. To avoid contamination, temporarily cap the sample tubes with 8-tube strip Axygen caps until they are seated in the work surface. 4. Place the sample on ice until ready to load onto the Apollo 324 work surface. IMPORTANT: The instrument is calibrated for Axygen tubes only. Using other types might result in run failure. Preparing Ligation Mix This recipe prepares ligation mix for one run of 32 samples. See Table 2-1 for ligation mix component volumes. 1. Prepare the ligation buffer and enzyme mix in a sterile 1.5 ml microfuge tube (for one run of 32 samples, plus four samples for volume loss to surfaces) as follows: a. Thaw, vortex, and briefly centrifuge the T4 DNA Ligase Buffer and Annealed Adapter tubes. b. Thaw and briefly centrifuge the T4 DNA Ligase Enzyme tubes. c. Combine 432 µl of T4 DNA Ligase Buffer, 36 µl of T4 DNA Ligase Enzyme, and 72 µl of Annealed Adapter set (5µM) in a 1.5 ml tube. Pipette mix and briefly centrifuge. Keep the mixture on ice. Table 2-1 Reagent volumes for ligation mix 32 Samples + 4 for pipetting loss 1 sample T4 DNA Ligase Buffer 432 µl 12 µl T4 DNA Ligase Enzyme 36 µl 1 µl Annealed Adapter Set (5µM) 72 µl 2 µl d. Aliquot 15 µl of this ligation mix into each tube of four 8-tube strips. NOTE: If you are adding individual adapters: Combine the T4 DNA Ligase Buffer and T4 DNA Ligase Enzyme. Aliquot 13 µl of this mixture into each tube of four 8-tube strips. Add 2 µl of adapter into each tube. 2. Temporarily cap the tubes and keep on ice. Repeat for each set of ligation mix tubes. NOTE: To maximize DNA product yield, use a heat sealer to seal the ligation mix tubes. Improper heat sealing or failure to heat seal the ligation mix could result in a decrease in yield in the range of 15 30%. CAUTION Heat sealing the ligation mix tubes requires the use of the IntegenX cold block for proper sealing. When using the cold block, be sure that the spacer posts are at the top of the cold block to ensure a proper seal. PrepX-32i DNA Library Protocol 7

12 Chapter 2 Preparing Samples and Reagents Preparing Ligation Mix 3. Heat seal the ligation mix tubes with aluminum heat seal, as follows: a. Turn on the heat sealer to pre-heat (approximately 10 minutes). If the temperature can be set, set it to 165 C. If the time can be set, set it between 0.5 to 1 second. b. After the plate sealer has pre-heated, retrieve the IntegenX 8-well cold block from a -20 C freezer. Place the strip tube with the ligation mix into the cold block. c. Place the aluminum heat seal on top of the tubes seated in the cold block (correct side up as indicated on the packaging) and place the cold block with the foil seal aligned atop the tubes into the heat sealer deck. d. Insert the cold block into the sealer and seal the tubes by applying the heat for between 0.5 to 1 second. A proper seal is verified by the appearance of embossed circles at the tops of the tubes. e. Let the seal cool for 30 seconds and then use a utility knife or razor to cut out the 8-tube strip with the ligation mix. f. Repeat for each set of ligation mix tubes. 4. Return the ligation mix to ice until ready to place onto the Apollo 324 work surface. Visually inspect the tubes to be sure that the entire volume is at the bottom of the tubes, without air pockets. 8 PrepX-32i DNA Library Protocol

13 Chapter 3 Setting Up and Running the Protocol Overview CAUTION In this chapter: Setting Up a Run Overview 9 Setting Up a Run 9 Running the Protocol 18 PCR and Post-PCR Conditions 19 Reagent and Reaction Locations 20 This chapter describes how to set up and run the protocol, providing guidance for placing the reagents, samples and consumables on the work surface, and then running the protocol. The work surface setup window in the touchscreen interface is mapped to the work surface blocks on the instrument. The window provides guidance for placing reagents, samples and consumables, and then running the protocol. The default setup of 32 samples fills the available blocks on the work surface. For details on preparing the samples and reagents before loading them in the instrument, see Chapter 2, Preparing Samples and Reagents. For details on placing: Consumables, see Placing Consumables on the Work Surface. Bead Mix strips, enzymes, samples and AMPure beads, see Loading Samples and Reagents. An accumulation of discarded pipettes and tips in the waste tip box can cause a run to fail. Be sure to open the waste tip box access door and check that the box has been emptied. This section describes how to load items on the instrument, using the setup window to verify placement. PrepX-32i DNA Library Protocol 9

14 Chapter 3 Setting Up and Running the Protocol Setting Up a Run Work Surface Layout The following illustration shows the layout of the work surface and placement of the consumables, reagents and samples for the protocol run. The default setup of 32 samples fills the available blocks on the work surface. The setup windows provide guidance for setting up runs. 1.1 ml Strip Tubes Piercing Tips Filter Tips ABCDE FGH A BCDE FGH Setup Window Launching the Software Block 1 Block 2 Block 3 Block A1 MICROPLATE H1 Reservoir 4 0.5M EDTA Reservoir 3 70% EtOH Reservoir 2 Empty Reservoir 1 H2O Block 5 Block 6 BeadX Strips Samples 1-8 Samples 9-16 Samples Samples Enzyme Strips ABCDE FGH ABC DE FGH The setup window in the touchscreen interface provides guidance for setting up runs. In the setup window, the NEXT and BACK buttons enable you to navigate so you can easily view the setup for any block. NEXT magnifies the next sequential block. BACK returns to the last magnified block or the Start window. For details on using the touchscreen interface, refer to the Apollo 324 User Guide. 1. Power on the instrument. The software start-up window is displayed for a few seconds, and then the IntegenX splash screen appears. When you power on the instrument, the temperature in the heating/cooling units (Blocks 3 and 4) adjusts to 18 C. After the splash screen disappears, the initial Start window is displayed, showing the available protocols. After the software is launched, the pipette head of the Apollo 324 System will initialize. IMPORTANT: Pressing in the center of the window or pressing and holding any of the buttons for several seconds will initiate calibration of the touchscreen. For details, see the Apollo 324 System User Guide. Filter Tips Block 7 Block 8 5X Beads 5X Beads Empty Tubes Product 1-8 Product 9-16 Product Product Ligase Mix 1-8 Ligase Mix 9-16 Ligase Mix Ligase Mix Filter Tips 10 PrepX-32i DNA Library Protocol

15 Setting Up a Run Chapter 3 Setting Up and Running the Protocol 2. To select the protocol, in the Start window, press PrepX-32i DNA Library. The work surface setup window is displayed. On the instrument work surface, the left-hand heating/cooling unit (Block 3) remains at 18 C, while the right-hand unit (Block 4) cools to 4 C. While Block 4 is cooling, the COOLING button is displayed. When Block 4 reaches 4 C, the RUN button appears. COOLING button displayed during cooling of Block 4 RUN button displayed when Block 4 reaches 4 C 3. While the unit is cooling, pre-load consumables, samples and reagents on the work surface. NOTE: You can place the consumables (tubes, plates, reservoirs and tips) on the work surface before starting the software and loading the samples and reagents. For details, see Placing Consumables on the Work Surface. For instructions on loading reagents and samples, see Loading Samples and Reagents. PrepX-32i DNA Library Protocol 11

16 Chapter 3 Setting Up and Running the Protocol Setting Up a Run Placing Consumables on the Work Surface Placing the consumables on the work surface can be done in advance. 1. Place ml tubes (eight each in 12-tube strips) in Block 1. Verify that they are properly seated and level. 2. Place a clean, empty 96-well microtiter plate in Block 2, with the A1 well in the lower left corner. 12 PrepX-32i DNA Library Protocol

17 Setting Up a Run Chapter 3 Setting Up and Running the Protocol 3. Place the piercing tips and dispensing filter tips into Block 5 as follows: a. Place 24 grey piercing tips into rows of Block 5. b. Place 72 dispensing tips into rows 1 9 of Block 5. NOTE: Do not use the plastic carrier tray in Block 5, as it might cause tips to stick during automatic operation. 4. Place four empty reservoirs in Block 6. You will fill these later. 5. Place 96 dispensing filter tips into Block Place 96 dispensing filter tips into Block Verify that the waste tip box is empty. PrepX-32i DNA Library Protocol 13

18 Chapter 3 Setting Up and Running the Protocol Setting Up a Run Loading Samples and Reagents 1. If you have not already done so, press the PrepX-32i DNA Library button in the Start window to initiate the protocol. The work surface setup window is displayed. On the instrument work surface, the left-hand heating/cooling unit (Block 3) remains at 18 C, while the right-hand unit (Block 4) cools to 4 C. While it is cooling, the COOLING button is displayed; when it reaches 4 C, the RUN button appears. While the unit is cooling, you can place samples and reagents on the work surface. IMPORTANT: Do not load the ligation mix until the Block 4 heating/cooling unit is at 4 C and the RUN button appears (approximately three minutes). 2. Load the prepared sample and reagents into Block 3 as follows: a. Place each of the eight Bead Mix 4-tube strips (black) vertically into rows 9 12 at the top of the block so that the arrows point up toward the rear of the work surface. b. Place 8-tube strips with the sample (tube caps removed) into rows 5, 6, 7 and 8. c. Place each of the eight 4-tube enzyme strips (orange) vertically into rows 1 4 at the bottom of the block so that the arrows point up toward the rear of the work surface. NOTE: Verify that all tubes are oriented and seated correctly. CAUTION Visually inspect the tubes as you place them in the block to ensure that the entire volume is in the bottom of the tubes, without droplets on the side walls, bubbles or void volume. If necessary, centrifuge briefly before placing the tubes. 14 PrepX-32i DNA Library Protocol

19 Setting Up a Run Chapter 3 Setting Up and Running the Protocol 3. Install the metal retention plate over Block 3 to secure the tubes and keep them stable, aligning the guide pins at the top and bottom. Rotate the side knobs to lock the retention plate in place. PrepX-32i DNA Library Protocol 15

20 Chapter 3 Setting Up and Running the Protocol Setting Up a Run 4. Load the prepared beads, empty product tubes and ligation mix into Block 4 as follows: a. Place two empty 8-tube strips for the 5X AMPure beads into rows 11 and 12. NOTE: Just before starting the protocol, you will aliquot the AMPure beads into these tubes (step 5). This is done later in order to avoid bead settling. For details on preparing the beads, see Preparing 5X AMPure XP Beads in Chapter 2. b. Place an empty 8-tube strip tube for resuspending the beads in row 10. c. Place four empty 8-tube strips for receiving products in rows 5, 6, 7 and 8. d. After the RUN button appears, place the four heat-sealed 8-tube strips with ligation mix into rows 1 through 4 of Block 4. NOTE: Verify that all tubes are oriented and seated correctly. 5. Load the AMPure beads into Block 4 as follows: a. Remove the two empty 8-tube strips from rows 11 and 12 in Block 4. b. Re-vortex the 5X AMPure beads. c. Aliquot 240 µl of 5X AMPure beads into each tube of each strip. d. Place the strips back in rows 11 and 12. NOTE: Do not centrifuge the bead strips before placing in row 11 and 12. Pipette out any air pockets in the tubes. 6. Install the metal retention plate over Block 4 to secure the tubes and keep them stable, aligning the guide pins at the top and bottom. Rotate the side knobs to lock the retention plate in place. 16 PrepX-32i DNA Library Protocol

21 Setting Up a Run Chapter 3 Setting Up and Running the Protocol 7. Fill the reagent reservoirs in Block 6 as follows: a. Dispense 25 ml of molecular biology grade water into Reservoir 1. b. Dispense 40 ml of freshly prepared 70% v/v EtOH into Reservoir 3. c. Dispense 15 ml of 0.5M EDTA into Reservoir 4. d. Leave Reservoir 2 empty, as it is used for waste. 8. Verify placement of all reagents and consumables and check that all tubes, plates and reservoirs are seated properly. Verify that all components are installed according to the setup window. You are now ready to start the run. For instructions, see Starting a Run. PrepX-32i DNA Library Protocol 17

22 Chapter 3 Setting Up and Running the Protocol Running the Protocol Running the Protocol Starting a Run When all of the items are on the work surface, close the instrument door. IMPORTANT: You must close the door in order to start the run. If you are ready to start the protocol, press RUN. If you are not ready, press NEXT, BACK or any block to review the setup and make changes to items on the work surface. Monitoring a Run After you press RUN, the front door of the instrument locks and the run starts. The status of each step of the run is displayed in a progress bar. After a run begins, the progress window shows the status of each step. A countdown timer shows how much time (minutes and seconds) is left for each step. From this window, you can stop a run completely. The STOP button appears only after a run has started. 18 PrepX-32i DNA Library Protocol

23 PCR and Post-PCR Conditions Chapter 3 Setting Up and Running the Protocol Finishing a Protocol After the products have been eluted off the beads, the protocol is finished. A message informs you when the protocol is complete. DANGER Press OK to display the Start window. At this point, the door unlocks. Do NOT attempt to open the instrument door until the run is finished. Do NOT assume that a protocol is complete until the PROTOCOL FINISHED message is displayed. Retrieving and Handling the Processed Libraries 1. Open the door and remove the 8-tube product strips (in rows 5, 6, 7 and 8, Block 4) from the instrument. 2. Cap the processed samples immediately and store them on ice or at -20 C. 3. Remove and discard used reagents and consumables. The DNA samples are now ready for amplification. PCR and Post-PCR Conditions IMPORTANT: To enrich the DNA libraries, we recommend using 15 cycles of PCR with 1 ng of sample DNA. For recommendations and instructions on PCR and post-pcr conditions, refer to the Apollo 324 System User Guide. PrepX-32i DNA Library Protocol 19

24 Chapter 3 Setting Up and Running the Protocol Reagent and Reaction Locations Reagent and Reaction Locations Block 1 Axygen 1.1 ml Tubes Block 2 Microtiter Plate Block ml PCR Tubes (PCR 0) Block ml PCR Tubes (PCR 1) Row Material Description Row Description Row Material Description Row Material 12 Empty n/a 12 AT reaction (1-8) 12 Bead 1B (130 µl) AT cleanup 12 5X beads (240 µl) 11 Empty n/a 11 AT reaction (9-16) 11 Bead 3 (90 µl) Low cut 11 5X beads (240 µl) 10 EtOH (70%) (1-8) ER bead wash samples AT reaction (17-24) 10 Bead 2 (130 µl) High cut 10 Resuspend beads 9 EtOH (70%) (9-16) ER bead wash samples AT reaction (25-32) 9 Bead 1A (130 µl) ER cleanup 9 8 EtOH (70%) (17-24) AT bead wash samples BeadX Sup (1-8) 8 Samples (1-8) (15 µl) ER Reaction 8 Product (1-8) 7 EtOH (70%) (25-32) AT bead wash samples BeadX Sup (9-16) 7 Samples (9-16) (15 µl) ER Reaction 7 Product (9-12) 6 Empty n/a 6 BeadX Sup (17-24) 6 Samples (17-24) (15 µl) ER Reaction 6 Product (17-24) 5 Empty n/a 5 BeadX Sup (25-32) 5 Samples (25-32) (15 µl) ER Reaction 5 Product (25-32) 4 Water Wash (1-8) Water elution samples Ligation reaction (1-8) 4 ER Enzyme (22 µl) End repair 4 Ligation Mix (1-8) (15 µl) 3 Water (9-16) Water elution samples Ligation reaction (9-16) 3 ER Buffer (55 µl) End repair 3 Ligation Mix (9-12) (15 µl) 2 Water (17-24) Water elution samples Ligation reaction (17-24) 2 AT Enzyme (22 µl) A-tail 2 Ligation Mix (17-24) (15 µl) 1 Water (25-32) Water elution samples Ligation reaction (25-32) 1 AT Buffer (55 µl) A-tail 1 Ligation Mix (25-32) (15 µl) Block 5 Tip Rack Block 6 Reservoirs Block 7 Tip 1 Row Material Description Material Row Material Description Row Material Description 12 Piercing tip Pierce bead solutions 12 Filter tip Ligation incubate (9-16) 12 Filter tip Ligation capture 2 (25-32) 11 Piercing tip Pierce enzymes and buffers EDTA (15 ml) 11 Filter tip Ligation incubate (1-8) 11 Filter tip Ligation capture 2 (17-24) 10 Piercing tip Pierce Ligation mix 10 Filter tip AT capture (25-32) 10 Filter tip Ligation capture 2 (9-16) 9 Filter tip 70% EtOH dispense 9 Filter tip AT capture (17-24) 9 Filter tip Ligation capture 2 (1-8) 8 Filter tip ER capture (1-8) 70% EtOH (40 ml) 8 Filter tip AT capture (9-16) 8 Filter tip Ligation capture 1 (25-32) 7 Filter tip ER reaction (25-32) 7 Filter tip AT capture (1-8) 7 Filter tip Ligation capture 1 (17-24) 6 Filter tip ER reaction (17-24) 6 Filter tip AT incubate (25-32) 6 Filter tip Ligation capture 1 (9-16) 5 Filter tip Water dispense and make Bead Stock 1B Empty 5 Filter tip AT incubate (17-24) 5 Filter tip Ligation capture 1 (1-8) 4 Filter tip ER reaction (9-16) 4 Filter tip AT incubate (9-16) 4 Filter tip Make Bead 3 3 Filter tip Make Bead Stock 1A 3 Filter tip ER capture (25-32) 3 Filter tip Bead Stock 2 prep and make Bead 2 2 Filter tip Mix ER and ER reaction (1-8) H 2 O (25 ml) 2 Filter tip ER capture (17-24) 2 Filter tip Ligation incubate (25-32) 1 Filter tip Mix AT and AT reaction (1-8) 1 Filter tip ER capture (9-16) 1 Filter tip Ligation incubate (17-24) Block 8 Tip 2 20 PrepX-32i DNA Library Protocol

25 Index A adapter mix, preparing 6 AMPure beads loading in tubes 16 placing tubes 16 preparing 6 B buffers placing 17 supplied 3 C consumables placing on the work surface 12 supplied by customer 4 D DNA libraries recommendations for PCR and cleanup 19 retrieving and handling 19 E enzymes placing tubes 14, 20 supplied 3 tube location 20 EtOH placing 17 preparing 70% 6 F filter tips location 20 placing 13, 20 H heating/cooling units, placing retention plates 15, 16 I instruments required for runs 4 L libraries, retrieving and handling 19 ligation mix heat sealing 8 placing tubes 16 preparing 7 M materials for operation customer-supplied reagents and consumables 4 instruments 4 reagent kit 3 microtiter plate, placing 12 molecular biology grade water placing 17 preparing aliquot 6 P PCR and post-pcr cleanup conditions 19 piercing tips location 20 placing 13, 20 pipette tips location 20 placing 13, 20 product placing tubes 16, 20 retrieving and handling 19 tube location 20 protocol overview 1 protocol runs ending 19 monitoring progress 18 performing 9 placing consumables 12 setting up 10 starting 18 PrepX-32i DNA Library Protocol 21

26 Index R reagent and reaction locations 20 reagent kit contents 3 reagent reservoirs filling 17 location 20 placing 13 reagents locations on work surface 20 placing tubes for dispensed reagents 12, 14 preparing 3 PrepX-32i DNA Library Kit 3 supplied by customer 4 retention plates, on heating/cooling units 15, 16 S samples handling processed samples 19 location 20 placing tubes 14, 20 preparing 3, 7 recommendations for PCR and cleanup 19 setting up and running the protocol 9 setup window, starting runs 18 software finishing the protocol run 19 monitoring progress 18 setup window 10 start window 10 starting runs 18 touchscreen interface 10 starting runs 18 T technical support, contacting ii tips locations 20 placing 13 touchscreen interface 10 training, obtaining information about ii W waste tip box 13 work surface reaction locations 20 reagent and tip layout 20 workflow processing schematic 2 steps 2 22 PrepX-32i DNA Library Protocol

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28 IntegenX Inc Stoneridge Drive, Suite 300 Pleasanton, CA Part Number P036873, Rev. A 4/2012