Identity Automation DNA Normalization and PowerPlex Setup Protocol for the Tecan Freedom EVO Workstation

Transcription

1 AUTOMATED PROTOCOL Identity Automation DNA Normalization and PowerPlex Setup Protocol for the Tecan Freedom EVO Workstation Revised 1/13 EP4

2 Identity Automation DNA Normalization and PowerPlex Setup Protocol for the Tecan Freedom EVO Workstation Description of the DNA Normalization and PowerPlex Setup Method for the Tecan Freedom EVO Workstation. All technical literature is available at: Visit the web site to verify that you are using the most current version of this Automated Protocol. Promega Technical Services if you have questions on use of this system: 1. Description.... Product Requirements Materials to be Supplied by the User Before You Begin A. Sample Considerations B. Preparation of Solutions Automated Processing Requirements for the Tecan Freedom EVO Workstation A. Instrumentation Requirements B. Additional Hardware, Labware and Consumables Required C. Tecan Freedom EVO Initial Deck Configuration Description of the Identity Automation DNA Normalization and PowerPlex Setup Method Important Considerations Automated Processing Requirements for Full Workflow on the Tecan Freedom EVO Workstation Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

3 1. Description This document describes the automated protocol for the Identity Automation DNA Normalization and PowerPlex Setup method on the Tecan Freedom EVO automated liquid-handling workstation. For additional information about Identity Automation methods for human identification applications, please visit: For troubleshooting PowerPlex chemistry issues, please refer to the appropriate PowerPlex Technical Manual. Note: All Promega Technical Manuals are available at: The automated method for DNA normalization and PowerPlex reaction setup is compatible with all Promega PowerPlex Systems. The automated method supports amplification of extracted DNA (Normalization Protocol), direct amplification of swabs extracted with SwabSolution Reagent (Swab Protocol), direct amplification of nonfta punches pretreated with PunchSolution Reagent (Punch Protocol) and direct amplification of FTA punches (FTA Protocol). In addition, the automated method enables multiple amplification plates to be prepared during a single automated method run, supporting 1 plates for the Normalization Protocol, 1 4 plates for the Swab Protocol and 1 9 plates for the Punch and FTA Protocols. For more information visit: Note: STR Normalization Manager Software is required for use of this automated method.. Product Requirements Product Size Cat.# STR Normalization Manager Software 3 CD-ROM DG18 Not for Medical Diagnostic Use. 3. Materials to be Supplied by the User Normalization Protocol TE 4 buffer [1mM Tris (ph 8.),.1mM EDTA] or Water, Amplification Grade (Cat.# DW991), for sample dilution Swab Protocol SwabSolution Kit (Cat.# DC871) Punch Protocol PunchSolution Kit (Cat.# DC971) Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

4 4. Before You Begin 4.A. Sample Considerations Samples must be in 96-well format (96-well plate or strip tubes). Amplification of Extracted DNA Centrifuge extracted DNA samples briefly to remove any air bubbles that might be present in the wells, as air bubbles can interfere with sample aspiration. If quantitation data are imported into the STR Normalization Manager Software, the well locations noted in your data file must match the sample well layout. Direct Amplification of DNA from Swabs If swab heads are present in the deep-well plate during automated sample transfer, be sure that the substrate is completely covered. The liquid handler will aspirate a small volume of swab extract from near the top of the liquid. If the disposable tips strike the swab head, the tips may fail to aspirate sample or become bent, causing the tips to crash into the amplification plate or cross-contaminate nontarget wells. Whole swabs should be cut or snapped off uniformly and close to the head to prevent these issues. Direct Amplification of DNA from Storage Card Punches Static may be problematic when adding punches to amplification plate wells. For FTA card punches, adding PCR amplification mix or amplification-grade water to the well before adding the punch may help alleviate static problems. For nonfta card punches, adding PunchSolution Reagent to the well before adding the punch during pretreatment may help alleviate static problems. 4.B. Preparation of Solutions PCR amplification reagents (primer pair mix, master mix, etc.) should be completely thawed and vortexed well prior to use. The automated method provides the option to robotically prepare PCR amplification mix at volumes less than 1.3ml in a 1.5ml microcentrifuge tube. Larger volumes of PCR amplification mix must be prepared manually and placed into an appropriate tube or trough. Manually prepared PCR amplification mix must be vortexed thoroughly (several 5- to 1-second pulses) prior to placing the mix on the worktable. Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

5 5. Automated Processing Requirements for the Tecan Freedom EVO Workstation Confirm that you have the required instrument and labware listed in Sections 5.A and 5.B for use of the Identity Automation DNA Normalization and PowerPlex Setup method on the Tecan Freedom EVO workstation. For automation of additional products, including full workflow automation, refer to Section 8 of this manual and the appropriate automated protocol for the chemistry of interest. 5.A. Instrumentation Requirements Minimum Installation Requirements The following is a list of Tecan parts and their corresponding part numbers that are required for the Identity Automation DNA Normalization and PowerPlex Setup method on a Tecan Freedom EVO Workstation. Note: A Freedom EVO 1 worktable provides sufficient space for Identity Automation DNA normalization and PowerPlex setup. Description Quantity Tecan Part Number Tecan Freedom EVO 1 instrument (or larger) configured with 4 or 8 5µl 1 Contact Tecan syringes and Disposable Tip LiHa Arm (Freedom EVOware Standard software) Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier DiTi Carrier, 4-Position a Microplate Carrier, 3 Position, Landscape (MP 3Pos) b Tube Carrier, 16mm, 16-Position (1 16) c a Alternatively, two DiTi Carriers, 3-Position (Part# 1613) can be used; at least 4 tip rack positions are required. b The number of worktable positions required for labware varies based on the protocol and the number of sample plates processed (Table 1). Be sure to install enough labware carriers to support the maximum number of plates per automated method run. c Optional: For manually prepared single-tube PCR amplification mix volumes of ml. Table 1. Labware Positions Required on the Worktable by Protocol and Number of Sample Plates. Number of Sample Plates Protocol that can be Processed Labware Positions Required Normalization Protocol 1 4 or 8 (4 per plate processed) Swab Protocol ( per plate processed) Punch Protocol (1 per plate processed) FTA Protocol (1 per plate processed) 4 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

6 5.B. Additional Hardware, Labware and Consumables Required The following additional items are required for the Identity Automation DNA Normalization and PowerPlex Setup method on a Tecan Freedom EVO workstation. Additional Hardware Required Hardware Supplier Cat.# Description Number Required Promega V161 Four-Position Tube Holder Promega A661 Heat Block Adapter 1 (Swab Protocol only) Promega V851 Plate Clamp 96 (for use with nonskirted plates and strip tubes) 1 (optional) a Promega V861 Plate Stand (for use with nonskirted plates and strip tubes) 1 (optional) a Tecan or Trough, Disposable, 1ml, Polypropylene Natural or Trough, Disposable, 1ml, Polypropylene Gray 3 b a The Plate Clamp 96 and Plate Stand are optional for securing nonskirted 96-well plates or MicroAmp Strip Tubes on the worktable. The Applied Biosystems MicroAmp 96-Well Base, Part Number N81-531, or the Tecan Plate Adapter PCR, Part Number 3386, also may be suitable. b A 1ml trough is required to hold each Four-Position Tube Holder and Disposable Trough, 5 ml, Low Dead Volume (if required). Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

7 5.B. Additional Hardware, Labware and Consumables Required (continued) Consumables Required Consumable Supplier Cat.# Description Tecan 369 µl LiHa Disposable Tips with Filter Tecan µl LiHa Disposable Tips with Filter Tecan or Trough, Disposable, 1ml, Polypropylene Natural or Trough, Disposable, 1ml, Polypropylene Gray Promega V ml Square-Well, V-Bottom Deep Well Plate Promega V6781.ml, Square-Well Deep Well Plate (for pretreatment with SwabSolution Reagent) User-selected User-selected User-selected User-selected 96-well PCR plate or strip tubes for PCR amplification 96-well PCR plate or strip tubes containing DNA samples 1.5ml microcentrifuge tube (for preparing single-tube PCR amplification mix volumes of <1.3ml) 7ml transport tubes (optional; for preparing single-tube PCR amplification mix volumes of ml) Tecan Disposable Trough, 5 ml, Low Dead Volume Number Required (Per Plate Processed) Normalization Protocol Swab Protocol Punch and FTA Protocols <¼ rack <¼ rack <¼ rack 1 3 racks 1 rack 1 rack a 1 a 1 a 1 a 1 a 1 a 1 a 1 a 1 a a Only one tube or reservoir type is required per run; the type depends on the PCR amplification mix volume and user choice. 6 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

8 5.C. Tecan Freedom EVO Initial Deck Configuration 5µl Tips 5µl Tips Sample Plate 5µl Tips Sample Plate Extracted DNA µl Tips Amplification Plate MA Figure 1. Tecan Freedom EVO initial deck configuration. Shown are the minimum requirements for installation of the DNA Normalization and PowerPlex Setup method. The layout shown supports extracted DNA normalization and is shown as an example only; the automated method can be adapted to any worktable layout as long as these hardware or equivalent are present. Note that additional labware positions are required to run the maximum number of plates for each protocol. Grid 1 Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier Site 1 (rear) Trough, Disposable, 1ml Site (middle) Trough, Disposable, 1ml, with Promega Four-Position Tube Holder Site 3 (front) Trough, Disposable, 1ml, with Promega Four-Position Tube Holder Grid 4 DiTi Carrier, 4-Position Position 1 (rear) 5µl LiHa Disposable Tips with Filter Position (midrear) 5µl LiHa Disposable Tips with Filter (as needed) Position 3 (midfront) 5µl LiHa Disposable Tips with Filter (as needed) Position 4 (front) µl LiHa Disposable Tips with Filter Grid 1 Microplate Carrier, 3 Position Site 1 (rear) Empty 1.1ml Square-Well, V-Bottom Deep Well Plate (first dilution plate) as required for sample dilution Site (middle) Empty 1.1ml Square-Well, V-Bottom Deep Well Plate (second dilution plate) as required for sample dilution Site 3 (front) PCR amplification plate or tubes Grid 16 Mounting Plate, Te-Shake 1-Position with additional Microplate Positions Position (middle) Strip tubes or 96-well plate containing samples Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

9 5.C. Tecan Freedom EVO Initial Deck Configuration (continued) Tube holder at Grid 1, Site : Tube holder at Grid 1, Site 3: PowerPlex System concentrated buffer or master mix 1 PowerPlex System concentrated primer mix 1 Taq DNA polymerase; Water, Amplification Grade; or AmpSolution Reagent (as required) 1 Integrate up to 4 different control samples. 146TA Empty 1.5ml microcentrifuge tube 1 If manually preparing PCR amplification mix, these positions will be empty. Optional: Manually prepared PCR amplification mix. Figure. Configuration of PowerPlex reagents and tubes in the Four-Position Tube Holders at Grid 1, Sites and 3. It is important to secure all open caps in the Four-Position Tube Holder so that they do not interfere with pipetting steps. The minimum volume for each reagent is determined by the number of samples processed. The instrument will prompt you to add the appropriate minimum volume of these reagents. Variable and Data Inputs A series of variables is used to set the state of the system and sample processing requirements at run time. These variables may be set and prompted based on specific laboratory needs. Used Tip Variables: Used_DiTi_Tips, Used_DiTi5_Tips You will be prompted to enter the number of tips that were used in the DiTi (Site 4) and first DiTi 5 (Site 1) tip racks at the start of the automated method. These variables are used within the EVOware script to set the first usable DiTi Positions for these racks. Manual_MM_Prep The Manual_MM_Prep variable determines whether the PCR amplification mix will be prepared by the Tecan Freedom EVO workstation ( False ) or prepared manually by the operator ( True ). When this variable is set to False the operator will be prompted to place the required reagents on the deck. When this variable is set to True the operator will be prompted to manually prepare the PCR amplification mix and place it on the deck. This variable is set during method installation. PCR amplification mix volumes of up to 1.3ml may be prepared robotically; larger volumes require manual preparation. 8 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

10 Optimize The Optimize variable allows the user to choose 5µl or µl disposable tips to dispense the PCR amplification mix. For PCR amplification mix dispense volumes of less than 1µl, the 5µl disposable tips will provide better volume delivery consistency, making them the better choice for normalization and STR amplification of extracted DNA samples. The larger capacity of the µl disposable tips provides significantly faster PCR amplification mix dispensing and shorter method run times. The µl tips provide excellent volume delivery consistency at 1µl and above but also may be suitable for reduced-volume direct-amplification protocols for which volume consistency is less important. Set the Optimize variable to Speed to force the use of µl tips for all volumes. Set the Optimize variable to Accuracy to force the use of 5µl tips for all volumes. Set the Optimize variable to Automatic to enable automatic tip selection based on PCR amplification mix dispense volume. The default tip selection always will be 5µl tips when tip changes are required for the Punch or FTA Protocol runs. FTA_Dispense The FTA_Dispense variable directs liquid handling during dispense of the PCR amplification mix for the FTA Protocol runs. Set this variable to Bottom if samples are to be punched into the plate after dispense of the PCR amplification mix; the PCR amplification mix will be contact dispensed to the bottom of each amplification well, and the same disposable tips will be used to dispense to all wells. Set this variable to Side if samples are already present in the wells; the PCR amplification mix will be dispensed above the well bottom with the tips in contact with the side of the amplification well to ensure reagent delivery but avoid contact with sample punches at the bottom of the well. Change_Tips_Each_AmpDispense The Change_Tips_Each_AmpDispense variable determines whether tips are changed during dispense of PCR amplification mix to amplification wells containing sample card punches. When this variable is set to True, disposable tips are changed between each well; when this variable is set to False, the same disposable tips are used to dispense to all wells. Changing tips is recommended when dispensing reagents to plates containing sample card punches. STR Normalization Manager Software The Promega STR Normalization Manager Software is the user interface for entering information about the samples that will be processed in the Identity Automation DNA Normalization and PowerPlex Setup method run. Information about sample number, sample well locations, dilution strategy, PCR amplification mix preparation and dispense, and incorporation of amplification controls are automatically exported for use by the Freedom EVOware script. On-site method installation by Promega includes training, installation and initial setup of the STR Normalization Manager Software for your laboratory. For more information visit: Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

11 6. Description of the Identity Automation DNA Normalization and PowerPlex Setup Method This overview describes the general liquid-handling steps required for the Identity Automation DNA Normalization and PowerPlex Setup method on the Tecan Freedom EVO workstation. Note: The Identity Automation DNA Normalization and PowerPlex Setup method does not provide automated transfer of SwabSolution Reagent or PunchSolution Reagent or on-deck heated incubation for sample preprocessing. 1. Diluent Transfer: The liquid handler transfers diluent to one or two dilution plates. The number of dilution plates used and the volumes transferred depend on the dilution required for the samples being processed. If no samples require dilution, this step will be skipped. Automation for direct amplification of storage card punches and swabs does not require diluent transfer or dilution plates.. Preparation of PCR Amplification Mix: If the user chooses to use the liquid handler to prepare the PCR amplification mix, the required volume of each reaction component will be transferred to a 1.5ml microcentrifuge tube and tip-mixed. Alternatively, manually prepared PCR amplification mix may be placed on the worktable in a 1.5ml tube, a 7ml transport vial or a 5ml low-dead-volume trough. 3. Dispense of PCR Amplification Mix: The liquid handler transfers the PCR amplification mix to appropriate wells of each amplification plate or set of strip tubes, dispensing by column from column 1 to column 1 for each set of 96 amplification wells. PCR amplification mix may be transferred using 5µl or µl tips depending on script settings and end-user input during installation. When possible, PCR amplification mix is aspirated in bulk and dispensed to multiple wells of the amplification plate to minimize run time. Programming is provided for changing tips between wells containing sample punches (Punch and FTA Protocols) to prevent cross-contamination. When working with multiple amplification plates, the workstation dispenses reagents in the order that the plates were configured in the STR Normalization Manager Software. 4. Sample and Transfer to the Amplification Plate. Extracted DNA Normalization Protocol: Samples and controls are processed by column from column 1 to column 1 based on the final amplification plate layout. The liquid handler processes each DNA sample and control through one of three dilution pathways to achieve the desired template amount in the final PCR amplification. Direct Transfer: Samples and diluent are aspirated separately and transferred directly to the amplification wells. One : Samples are diluted through a single dilution plate; up to a 1-fold dilution can be achieved. Two : Samples are diluted through two dilution plates; up to 1-fold dilution can be achieved in each dilution plate for a maximum 1,-fold dilution. Table shows how each dilution pathway achieves the desired template amount in each PCR amplification reaction. Swab Protocol: Extracts prepared using the SwabSolution Reagent and controls are processed by column from column 1 to column 1 based on the final amplification plate layout. The specified volume of each sample and control is aspirated and dispensed to the appropriate amplification well. If Water, Amplification Grade, is not included in the PCR amplification mix, the required volume will be prompted during worktable setup and aspirated prior to aspirating each sample or control. Punch and FTA Protocols: Controls are processed by column from column 1 to column 1 based on the final amplification plate layout. The specified volume of each control is aspirated and dispensed to the appropriate amplification well. 1 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

12 Table. Direct-Transfer, One- and Two- Strategies Applied Across a Range of Starting Sample s. All volumes are in microliters (µl). Sample Concentration (ng/µl) ,,5 5, Pathway Direct Transfer Direct Transfer Direct Transfer One One Two Two Two Two Two 6, Two Volume into First Volume of Diluent into First Volume into Second Volume of Diluent into Second Volume into Final Plate Volume of Diluent into Final Plate Template DNA Amplified (ng) Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

13 7. Important Considerations 1. Always use aerosol-resistant tips to minimize the risk of cross-contamination.. All PowerPlex System reagents must be thoroughly mixed by vortexing before placing them on the deck for a run. This includes manually prepared PCR amplification mix containing DNA polymerase; vigorous mixing will ensure homogeneity and will not harm performance. Mix as directed in the appropriate PowerPlex System Technical Manual. 3. The calculations for PCR amplification mix preparation include excess reagent to ensure that enough PCR amplification mix is prepared for all amplification wells. 4. Liquid Classes must be calibrated to ensure accurate volume handling for both samples and amplification reagents. Calibration checks are performed as part of Promega standard installation service. 5. The Promega STR Normalization Manager Software has been integrated into methods for the Identity Automation DNA Normalization and PowerPlex Setup on the Tecan Freedom EVO, Beckman Coulter Biomek 3, Biomek NX P and Biomek FX P platforms. For more information or to inquire about the potential to integrate this software onto other instruments, please visit: 1 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

14 8. Automated Processing Requirements for Full Workflow on the Tecan Freedom EVO Workstation Full Workflow Requirements The following is a list of Tecan parts and their corresponding part numbers that are required for the automated Differex System, DNA IQ System, Plexor HY System and DNA Normalization and PowerPlex Setup methods on a Tecan Freedom EVO workstation. A Freedom EVO 1 or 15 worktable is required for full workflow automation; please contact your local sales representative for more information. Description Quantity Tecan Part Number Tecan Freedom EVO 1 instrument (or larger) configured with 4 or 8 5µl syringes, disposable Tip LiHa Arm and ROMA Gripper (Freedom EVOware Standard software) 1 Contact Tecan Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier a DiTi Carrier, 4-Position (for re-racking DiTi tips) a Shelf, 16-Position (4 sites with 4 shelves per site) a Microplate Carrier, 3-Position, Landscape (MP 3Pos) or alternative carrier(s) to provide up to 9 microplate positions Trough/DiTi Carrier, 1ml, 3-Position, with 3 dedicated positions for reusing disposable tips a Tube Carrier, 1.5ml or.ml Microcentrifuge with Hinged Cap, 16-Position a Te-Shake Microplate Shaker Unit a Microplate Nest, 1-Position, with Hold Down, Te-Shake Shaker a Mounting Plate, Te-Shake 1-Position with additional Microplate Positions a EchoTherm Dry Bath, Torrey Pines Scientific, IC, Electronic Chilling/Heating (or equivalent; contact Tecan for additional options on heating units, adaptors and mounting hardware) a Microplate Adaptor, Torrey Pines Scientific, IC/IC/IC5 a Mounting Plate, Torrey Pines Scientific EchoTherm Dry Bath, IC/IC/IC5 a a These parts are included in the Tecan Package #16. Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

15 8. Automated Processing Requirements for Full Workflow on the Tecan Freedom EVO Workstation (continued) Additional Hardware, Labware, and Consumables Required The following additional items are required for Identity Automation full workflow processing on a Tecan Freedom EVO workstation. Additional Promega Hardware Required for Full Workflow (Differential Extraction, DNA Purification, DNA Quantitation, Normalization and STR Analysis) Number Required for the Indicated Automated Method Cat.# Description Differex Method DNA IQ Method Plexor HY Method PowerPlex Normalization V641 MagnaBot Flat Top Magnetic Separation Device 1 A661 Heat Block Adapter 1 V8151 MagnaBot 96 Magnetic Separation Device 1 Z331 1/4 inch Foam Spacer 1 V6741 Deep Well Heat Transfer Block 1 V161 Four-Position Tube Holder V851 Plate Clamp 96 (for use with nonskirted plates and strip tubes) 1 (optional) a 1 (optional) a V861 Plate Stand (for use with nonskirted plates and strip tubes) 1 1 (optional) a (optional) a a The Plate Clamp 96 and Plate Stand are optional for securing nonskirted 96-well plates or MicroAmp Strip Tubes on the worktable. The Applied Biosystems MicroAmp 96-Well Base, Part Number N81-531, or the Tecan Plate Adapter PCR, Part Number 3386, also may be suitable. 14 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

16 Additional Consumables Required for Full Workflow Automation (Differential Extraction, DNA Purification, DNA Quantitation, Normalization and STR Analysis) Supplier Cat.# Description Number Required (Per Plate Processed) for the Indicated Automated Method(s) Differex Method DNA IQ Method Plexor HY Method PowerPlex Normalization Tecan µl LiHa Disposable Tips with Filter <¼ rack <¼ rack Tecan 369 µl LiHa Disposable Tips with Filter 1 rack racks <¼ rack <¼ rack Tecan µl LiHa Disposable Tips with Filter 1 rack 1 3 racks Tecan Trough, Disposable, 1ml, Polypropylene 3 1 Gray Tecan Disposable Trough, 5 ml, Low Dead Volume 1 a Promega V6791 Pyramid-Bottom Reservoir, 1 Column 1 b 1 b Promega V ml, Round-Bottom Deep Well Plate 1 4 Promega V6781.ml, Square-Well Deep Well Plate 1 c Promega V1391 Slicprep 96 Device 1 1 c Promega V ml, Square-Well, V-Bottom Deep Well Plate User-selected 96-well PCR plate or strip tubes for PCR amplification User-selected 96-well PCR plate or strip tubes for Plexor HY amplification User-selected 96-well PCR plate or strip tubes for standard curve preparation User-selected 96-well PCR plate or strip tubes for purified DNA samples User-selected 1.5ml microcentrifuge tube 1 a User-selected 7ml transport tubes (optional; for preparing single-tube PCR amplification mix volumes of ml) a Only one tube or reservoir type is required per run; the type depends on the PCR amplification mix volume and user choice. b The same reservoir can be used for the Differex and DNA IQ methods. c The.ml, Square Well Plate or SlicPrep 96 Device may be used for samples; two plates are required under the hanging µl pipette tips. Samples processed using the Differex System do not require an additional plate a 1 a Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

17 8. Automated Processing Requirements for Full Workflow on the Tecan Freedom EVO Workstation (continued) 5µl Tips 5µl Tips Sample Plate 5µl Tips Sample Plate Extracted DNA µl Tips Amplification Plate MA Figure 3. Tecan Freedom EVO initial deck configuration. Shown is a worktable that supports the full suite of Promega automated methods for the Tecan Freedom EVO workstation (Differex System, DNA IQ System, Plexor HY System and DNA Normalization and PowerPlex Setup). The labware supports extracted DNA normalization and amplification and is shown as an example only; the automated method can be adapted to any worktable layout as long as these hardware or equivalents are present. Grid 1 Trough/DiTi Carrier, 1ml, 3-Position, with associated tip rack positions Position 1 (rear) Trough, Disposable, 1ml Position (middle) Trough, Disposable, 1ml, with Four-Position Tube Holder Position 3 (front) Trough, Disposable, 1ml, with Four-Position Tube Holder Grid 3 DiTi Carrier, 4-Position Position 1 (rear) 5µl LiHa Disposable Tips with Filter Position (midrear) 5µl LiHa Disposable Tips with Filter (as needed) Position 3 (midfront) 5µl LiHa Disposable Tips with Filter (as needed) Position 4 (front) µl LiHa Disposable Tips with Filter Grid 9 Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier Grid 1 17 Shelf, 16-Position (if starting samples are in tubes) Positions ml microcentrifuge tubes containing samples Note: Be sure to turn all open caps so that the caps do not interfere with sample aspiration. Grid 18 Microplate Carrier, 3-Position Site 1 (rear) Empty 1.1ml Square-Well, V-Bottom Deep Well Plate (first dilution plate) as required for sample dilution Site (middle) Empty 1.1ml Square-Well, V-Bottom Deep Well Plate (second dilution plate) as required for sample dilution Site 3 (front) Amplification plate or tubes Grid 4 Mounting Plate, Te-Shake 1-Position with additional Microplate Positions Position (middle) Strip tubes or 96-well plate containing samples (extracted DNA) Grid 33 EchoTherm Dry Bath, Torrey Pines Scientific, IC, Electronic Chilling/Heating, with Promega Deep-Well Heat Transfer Block (used with the DNA IQ System only) 16 Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13

18 11 13 Promega Corpora on. All Rights Reserved. MagnaBot, Plexor and PowerPlex are registered trademarks of Promega Corpora on. AmpSolu on, Differex, DNA IQ, Iden ty Automa on, PunchSolu on, Slicprep, STR Normaliza on Manager and SwabSolu on are trademarks of Promega Corpora on. Biomek is a registered trademark of Beckman Coulter, Inc. Freedom EVO and EVOware are registered trademarks of Tecan AG Corpora on. FTA is a registered trademark of Flinders Technologies, Pty, Ltd., and is licensed to Whatman. MicroAmp is a registered trademark of Applera Corpora on. Te-Shake is a trademark of Tecan AG Corpora on. Products may be covered by pending or issued patents or may have certain limita ons. Please visit our Web site for more informa on. All prices and specifica ons are subject to change without prior no ce. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date informa on on Promega products. Promega Corpora on 8 Woods Hollow Road Madison, WI USA Toll Free in USA Fax EP4 Revised 1/13