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Installation Guide for Genetic Analysis models including: S8-044 A1 Genomic DNA Extraction System, S8-044 A1, Genomic DNA Extraction System, DNA Extraction System, Extraction System
Resources | GA-map Dysbiosis Test
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DocumentDocumentINSTALLATION GUIDE for GA-map® Dysbiosis Test Lx v2 TABLE OF CONTENTS Overview ..................................................................................................................................................................2 Installation check list................................................................................................................................................2 Recommended safety equipment............................................................................................................................2 Computer requirements ..........................................................................................................................................2 Laboratory requirements .........................................................................................................................................3 Equipment, materials, and reagents required .........................................................................................................4 Training Plan.............................................................................................................................................................9 Contact info..............................................................................................................................................................9 OVERVIEW This guide provides the information required to prepare a laboratory for performing the GA-map® Dysbiosis Test Lx v2 assay. Requirements for instruments, materials, and reagents are described. INSTALLATION CHECK LIST The following installation checklist summarizes the elements that must be completed during the installation phase. Mark each item as it is completed. Once these prerequisites have been met, please contact Genetic Analysis AS to schedule the training. Instruments and consumable items for Step 1 Genomic DNA extraction (see tables on page 4-6) must be acquired Instruments and consumable items for Step 2 Amplification of the bacterial 16S rRNA gene Step 6 Hybridization and Signal Detection (see tables on page 6-9) must be acquired All instruments, including the Luminex detection system, must be installed and ready for use Operators must have been trained in use and maintenance of the Luminex detection system RECOMMENDED SAFETY EQUIPMENT Fecal samples should be treated as potentially infectious material until inactivation and require the use of BSL-2 grade laboratory equipment and precautions. This involves the use of appropriate PPE, biological safety cabinet, proper waste disposal and risk-minimizing routines for sample handling. Make sure to follow laboratory and local guidelines for EHS. COMPUTER REQUIREMENTS The GA-map® Analyzer is a cloud-based software compatible with most web browsers. See the GA-map® Analyzer Manual for details. S8-044 A1 v9 Appendix 6 Page 2 of 9 LABORATORY REQUIREMENTS The GA-map® Dysbiosis Test Lx v2 requires a typical molecular laboratory set-up. It is recommended that the laboratory contains, at a minimum, dedicated pre-PCR, template-free, and PCR/post-PCR zones. The zones should be separated to prevent contamination. The table below contains an overview of the different laboratory procedures that are performed in the individual zones, in addition to instruments and equipment needed in the different workstations. Please refer to the Equipment, materials, and reagents required section for equipment specifications. To equip the lab with appropriate pipettes, please refer to the IFU for volumes to be pipetted. Zone Lab procedures Workstation Pre-PCR Templatefree PCR/Post-PCR Sample preparation and gDNA extraction. Addition of template for the amplification step. Mastermix preparation for the amplification, clean-up, and End-Labeling steps. Amplification reaction. The quantification step. Addition of template for the End-labeling step and the End-labeling reaction. The Hybridization and signal detection step. Biological safety cabinet equipped with: · Vortex mixer · Decapper for Lysing Matrix-E tubes · Appropriate single- and 8-channel pipettes w/ wide orifice tips Open bench equipped with: · Bead beater · Plate centrifuge for Lysing Matrix-E tubes · Water bath · DNA extraction robot · Vortex mixer · Microcentrifuge · Dispenser pipette w/ tips · Single- and 8-channel pipettes with tips · Ice or cooling elements for other reagents and plate PCR cabinet with available equipment: · Vortex mixer · Microcentrifuge · Dispenser pipette w/ tips · Single channel pipettes w/ tips · Freezing block for enzymes · Ice or cooling elements for reagents and plate Open bench equipped with: · Plate centrifuge for quick spin · Thermal cycler · DNA quantification system · Ring magnet plate · Luminex detection platform · Vortex mixer · Microcentrifuge · Dispenser pipette w/ tips · Single- and 8-channel pipettes w/ tips · Ice or cooling elements for reagents and plate · Freezing block for plates S8-044 A1 v9 Appendix 6 Page 3 of 9 EQUIPMENT, MATERIALS, AND REAGENTS REQUIRED The lists below describe the required equipment, materials, and reagents for the GA-map® Dysbiosis Test Lx v2. All items are required, unless otherwise specified. Genetic Analysis must be informed of any deviations from these requirements and a separate validation might be necessary. The quantity for all equipment is based on the set-up described in the Laboratory Requirements section above. It should be noted that different laboratory set-ups (e.g. with additional zones) might affect the quantity. FOR STEP 1 GENOMIC DNA EXTRACTION List of equipment when extracting DNA according to STEP 1 of the IFU need of equipment may vary with choice of extraction kit and instruments. Equipment Specifications Recommended options type Decapper for Capacity: 8-12-channel 8-Channel Screw Cap Decapper Lysing Matrix-E capper/decapper for matrix (4105MAT), Thermo Scientific tubes tubes AlteCapTM Switch (404000) with Cassette 12/96 - Internal thread Matrix screw caps (404014), AltemisLab Bead beater Capacity: 96-well rack Force: 1600-1800 RPM FastPrep-96TM Homogenizer w/96well plate insert (116010500), MP Biomedicals Plate centrifuge Capacity: 96 well plate with Any min 6 cm height Force: 1300 rcf Water bath Capacity: 96 well plate Any Temperature: 65°C DNA extraction 96 well plate format KingFisher Flex 96 deep well head robot Magnetic particle processor (540 0630) w/magnetic micro-plate Separator, installed with KF Flex 96 KF heating block (2407 5420), Thermo Scientific Bioer GenePure (NAP-32P) Contact Genetic Analysis for approved instruments. Or request our standardized extraction validation procedure. Vortex mixer Speed: ~2800 rpm Any Micro Centrifuge Capacity: 1.5ml/ 2ml tubes Any (spin down only) S8-044 A1 v9 Appendix 6 Page 4 of 9 Dispenser Volumes to be dispensed: 20, Any pipette w/tips 200, 250, 270 and 720 µl Multipette®E3/E3x (4987000371/4987000380), Eppendorf Pipettes single Volumes: 10 1200 µl channel w/tips Wide orifice tips recommended for pipetting of fecal samples prior to gDNA extraction Pipettes 8- Volumes: 100 1200 µl channel w/tips Wide orifice tips recommended for pipetting of fecal samples prior to gDNA extraction Ice or cooling For keeping reagents/sample Any blocks intermediates cold during handling List of materials Material Specifications Lysing Matrix-E Capacity: 2 ml tubes Bead type: 1.4 mm Ceramic Spheres, 0.1 mm Silica Spheres, and 4 mm Glass Bead. Tube for Lysis Volume: 5, 15 or 50 ml mix Deep well plate DeepWell 96 Plate, V- for DNA bottom extraction robot Recommended options Lysing Matrix E, 96-tube rack, barcoded tubes, 1 Rack (116984001B), (note: individual Matrix tubes with screw cap), MP Biomedicals Any KingFisher Deepwell 96 Plate, Vbottom, (95040450), Thermo Scientific 96 Well Plate SW 2ml with V-Bottom PP (BAKR43001-0504), VWR Deep well Tip 96 tip comb for DeepWell KingFisher 96 tip comb for DW Combs for DNA magnets magnets, (97002534), Thermo extraction robot Scientific 96 Tip Comb PP (BAKR43001-0505), VWR Elution plate for 96 well microplate, 0.2 ml KingFisher 96 KF microplate (200 l), DNA extraction (97002540), Thermo Scientific robot Well Plate 96 SW 0.2ml with V- Bottom PP (BAKR43001-0506), VWR Adhesive PCR Suitable for deep well plate Adhesive PCR Plate Seals (AB0558), plate seal for DNA extraction robot Thermo Scientific Microtiter sealing tape Suitable for plates for DNA extraction robot Adhesive Plate Seals (AB0580), Thermo Scientific Microtiter plate Capacity: 96-well, 250 µl Any w/seal for gDNA dilution S8-044 A1 v9 Appendix 6 Page 5 of 9 List of reagents Reagent Specifications Extraction Fecal sample of known control positive quality (optional) DNA extraction Validated for GA-map® reagent kit Dysbiosis Test Lx v2 Ethanol (for 96-100% ethanol extraction kit) 500 ml Acetone (for 99% acetone extraction kit) 350 ml Water for dilution of gDNA and as PCR ctrl neg. Sterile, Nuclease-free water 500 ml Recommended options Any magTM maxi DNA purification kit, 288 tests (NAP40430), LGC Genomics Any Any Any FOR STEP 2 AMPLIFICATION OF THE BACTERIAL 16S RRNA GENE TO STEP 6 HYBRIDIZATION AND SIGNAL DETECTION List of equipment Equipment type Specifications Recommended options Used in assay steps Plate centrifuge Capacity: 96-well plate Any (quick spin down only) Step 2 Step 3 Step 4 Step 5 Step 6 Thermal cycler Capacity: 96-well VeritiTM 96-Well Thermal Cycler Step 2 Lower maximum volume (4375786), Applied Biosystems Step 4 75µl VeritiProTM 96-Well Thermal Cycler Temperature range: 4-105°C (A48141), Applied Biosystems Step 5 Step 6 T100TM Thermal Cycler (186-1096), Bio-Rad DNA System for quantifying quantification dsDNA (0-100ng/µl) system Qubit® Fluorometer 3.0 (Q33216) or 4.0 (Q33238) or Flex (Q33327), Invitrogen Step 3 Ring Magnet Plate Luminex detection platform Capacity: 96-well Hybridization plate, 100µl Luminex xMAP® technology Capacity: 96-well Multiplexing capacity: 50 FLUOstar OMEGA Microplate Reader with filter-based absorbance (415103) w/ appropriate filters for the quantification assay, BMG LabTech 96-Well Ring Magnet Plate (S380), Permagen MAGPIX® system or NxTAG®Enabled MAGPIX® system with xPONENT® version 4.2 or higher (MAGPIXXPON4.1-CEIVD), Luminex* Step 6 Step 6 S8-044 A1 v9 Appendix 6 Page 6 of 9 Laser type: Green and red Luminex® 200TM system with xPONENT® version 4.2 or higher (LX200-XPON-IVD/RUO), Luminex* Vortex mixer Speed: ~2800 rpm Any Step 2 Step 3 Step 4 Step 5 Step 6 Micro Capacity: 2ml tubes Any Centrifuge (spin down only) Step 2 Step 3 Step 4 Step 5 Step 6 Dispenser Volumes to be dispensed: Multipette®E3/E3x pipette w/tips 20µl and 40µl (4987000371/4987000380), Eppendorf Step 2 Step 5 Step 6 Pipettes single Volumes: 0.5 1000µl Any channel w/tips Step 2 Step 3 Step 4 Step 5 Step 6 Pipettes 8- Volumes: 0.5 300µl Any channel w/tips Step 2 Step 3 Step 4 Step 5 Step 6 Ice or cooling For keeping Any blocks reagents/sample intermediates cold during handling Step 2 Step 3 Step 4 Step 5 Step 6 Freezing block Capacity: 1.5/ 2ml reagent Benchtop cooler (5115-0012), for tubes tubes Thermo Scientific For use during handling of enzymes Step 2 Step 4 Step 5 Freezing block Capacity: 96-well microtiter PCR-Cooler 0.2ml (3881000031), for plates plate Eppendorf Step 5 For use during End-labeling *Additional items for use and maintenance are required: kits for calibration and performance verification, sheath fluid or drive fluid, and an ultrasonic bath (40-60 kHz) S8-044 A1 v9 Appendix 6 Page 7 of 9 List of materials Material Specifications Microcentrifuge Volume: 1.5, 2 and 5ml tubes for mixing of reagents Microtiter plate Capacity: 96-well for PCR/End- PCR grade labeling Recommended options Safe-Lock tubes, Eppendorf 96-Well Semi-skirted Flat Deck PCR Plates (AB1400), Thermo Scientific Used in assay steps Step 2 Step 4 Step 5 Step 6 Step 2 Step 5 96-Well Semi-skirted, segmented PCR Plate (AB0900), Thermo Scientific 8-cap sealing Suitable for microtiter plate Flat PCR Caps, strips of 8 (AB0784), Step 2 strips Thermo Scientific Step 5 Microtiter Suitable for microtiter plate Adhesive Plate Seals (AB0580), Step 2 sealing tape Thermo Scientific Step 5 Tubes/plates Suitable for the selected DNA For use with plate reader: NuncTM 96- Step 3 for DNA quantification system Well Microplate, Black (237108), quantification Thermo Scientific For use with Qubit® 3.0/4.0 Fluorometer: QubitTM Assay Tubes (Q32856), Invitrogen For use with Qubit® Flex Fluorometer: QubitTM Flex Assay Tube Strips (Q33252), Invitrogen Hybridization Capacity: 96-well, unskirted, 96-well Twin.tecTM PCR Plates, Step 6 plate 0.2ml Unskirted, Divisible (0030133358), Suitable for Luminex Eppendorf detection platform ThermowellTM 96-Well Polycarbonate PCR Microplates, Model P (6509), Corning Sealing film for Non-adhesive Microseal® 'A' (MSA5001), Bio-Rad Step 6 Hybridization plate For 96-well format Reagent 25ml Any Step 4 reservoir Step 6 List of reagents Reagent Specifications Recommended options Used in assay steps Water for PCR Sterile, Nuclease-free water, Any control neg. same as used for dilution of gDNA Step 2 Assay kit for For plate reader DNA quantification Quant-iTTM PicoGreenTM dsDNA Assay Kit (P11496), ThermoFisher Quant-iTTM 1X dsDNA HS Assay (Q33232), ThermoFisher Step 3 S8-044 A1 v9 Appendix 6 Page 8 of 9 For Qubit® Fluorometer QubitTM dsDNA HS assay kit (Q32854), ThermoFisher QubitTM 1X dsDNA HS assay kit (Q33231), ThermoFisher Quant-iTTM 1X dsDNA HS Assay (Q33232), ThermoFisher TRAINING PLAN Training will be scheduled following system set-up. Genetic Analysis will train users in performing the process of GA-map® Dysbiosis Test Lx v2 from fecal gDNA extraction (or from PCR, if the extraction method differs from that described in the IFU) to generation of the reports. Other sampling devices, extraction instruments and chemistry can be validated upon request using our standardized validation method. Basic instrument operation and use of the software will be included in the training. Those being trained will be required to have a basic knowledge of Microsoft Windows and use of general laboratory equipment and tools. Operators must have been trained in use and maintenance of the Luminex system prior to the tech transfer. CONTACT INFO We are happy to help you with your inquiries. Technical Support: support@genetic-analysis.com Genetic Analysis AS | Ulvenveien 80 | 0581 OSLO | Norway www.genetic-analysis.com | Phone: +47 48 32 16 10 | E-mail: info@genetic-analysis.com Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. All trademarks and registered trademarks mentioned herein are the property of their respective owners. © 2021-2023 Genetic Analysis, all rights reserved. S8-044 A1 v9 Appendix 6 Page 9 of 9