Identity Automation DNA IQ System Protocol for the Tecan Freedom EVO Workstation Automated Protocol #EP039

Transcription

1 Identity Automation DNA IQ System Protocol for the Tecan Freedom EVO Workstation Automated Protocol #EP039 DESCRIPTION OF THE DNA IQ SYSTEM METHOD FOR THE TECAN FREEDOM EVO WORKSTATION. All technical literature is available on the Internet at: Please visit the web site to verify that you are using the most current version of this Automated Protocol. 1. Description Requirements and Product Storage Conditions Materials to be Supplied by the User Before You Begin...2 A. Preparation of Solutions...2 B. Sample Processing Before Automated Processing (As Required) Automated Processing Requirements for the Freedom EVO Workstation...3 A. Instrumentation Requirements for the Identity Automation DNA IQ System Method on the Freedom EVO Workstation...3 B. Labware Requirements for the Identity Automation DNA IQ System Method on the Freedom EVO Workstation...5 C. Freedom EVO Workstation Initial Deck Configuration...6 D. Freedom EVO Workstation Variable Inputs...7 E. Freedom EVO Workstation Reagent Dispense Volumes Description of the Identity Automation DNA IQ System Method Important Considerations Description This document describes the automated protocol for the Identity Automation DNA IQ System(a) on the Tecan Freedom EVO laboratory automation workstation. This method is optimized to produce inhibitor-free DNA eluates even when very small elution volumes are used and a maximum volume of low-level DNA template is added to PowerPlex system reactions. Testing has shown successful amplification with 19.2µl of eluate using the PowerPlex 16 System (Cat.# DC6530 and DC6531). Please contact the Promega Genetic Identity Team (genetic@promega.com) prior to implementing this method on your workstation. To obtain information about Identity Automation methods for human identification applications, visit: Note: All Promega Technical Manuals are available at: Revised 3/12 Page 1

2 2. Requirements and Product Storage Conditions Product Size Cat.# DNA IQ System 100 reactions* DC reactions* DC6700 *Cat.# DC6701 contains sufficient reagents to process one 96-well plate of aqueous samples (up to 100µl volume) or lysis samples (samples on solid supports; up to 200µl volume). Cat.# DC6700 contains sufficient reagents for four 96-well plates at these volumes. When processing larger sample volumes, you will need to purchase additional Lysis Buffer (Cat.# A8261). Processing four 96-well plates of aqueous or lysis samples of 400µl volume, the maximum volume supported, requires an additional 70ml of Lysis Buffer. Storage Conditions: Store all components at room temperature (15 30 C). Items Available Separately Product Size Cat.# DNA IQ Resin 50ml A8251 Lysis Buffer 150ml A8261 2X Wash Buffer 70ml A8271 Elution Buffer 50ml A8281 Not for Medical Diagnostic Use. 3. Materials to be Supplied by the User DNA IQ System (If you are processing more than 100µl of each aqueous sample or 200µl of each lysis sample, you will need to purchase additional Lysis Buffer; see Section 2.) Slicprep 96 Device (Cat.# V1391) or 1.5ml microcentrifuge tubes and DNA IQ Spin Baskets (Cat.# V1221) for sample preprocessing as required (see Section 4.B) 99% isopropyl alcohol % ethanol DTT (Cat.# V3151 for 5g, Cat.# V3155 for 25g) See Sections 5.A and 5.B for instrumentation requirements and labware requirements, respectively. 4. Before You Begin 4.A. Preparation of Solutions Prior to beginning the Identity Automation DNA IQ System method, prepare the following solutions: Prepared Lysis Buffer 1. Add 1µl of 1M DTT for every 100µl of Lysis Buffer. 2. Mix by inversion several times. 3. Mark and date label to record addition of DTT. This solution can be stored at room temperature for up to one month if the bottle is tightly closed. Page 2 Revised 3/12

3 DNA IQ Resin Solution (prepared Lysis Buffer + Resin) Prepared Lysis Buffer + Resin is not required for processing differential extraction samples using the Differex System. 1. Thoroughly mix the DNA IQ Resin by inversion until the particles are completely resuspended. 2. Make the prepared Lysis Buffer as described above (i.e., add 1µl of 1M DTT for every 100µl of Lysis Buffer). 3. Prepare the DNA IQ Resin Solution by combining prepared Lysis Buffer and DNA IQ Resin at the volumes instructed by the DNA IQ Graphical User Interface (GUI); see Section 5.D. Note: Prepare the DNA IQ Resin Solution fresh before each run. Do not store. 4. Mix thoroughly by gentle inversion several times. To prevent foaming of the solution, do not mix vigorously. 1X Wash Buffer 1. Add ethanol and isopropyl alcohol directly to the 2X Wash Buffer (15ml of % ethanol and 15ml of 99% isopropyl alcohol for DC6701; 35ml of % ethanol and 35ml of 99% isopropyl alcohol for DC6700). 2. Replace the cap, and mix by inversion several times. 3. Mark label as 1X Wash Buffer, and indicate addition of alcohols. 4. Store at room temperature (15 30 C). Make sure bottle is closed tightly to prevent evaporation. 4.B. Sample Processing Before Automated Processing (As Required) For samples on solid supports and differential extraction samples, preprocessing must be performed prior to the start of the automated method. For more information about preprocessing samples on solid supports, refer to the DNA IQ System Small Sample Casework Protocol Technical Bulletin #TB296 or DNA IQ System Database Protocol Technical Bulletin #TB297, or contact Promega Technical Services. For preprocessing of differential extraction samples, refer to the Automated Differex System Protocol for the Tecan Freedom EVO System Automated Protocol #EP030, or contact Promega Technical Services. 5. Automated Processing Requirements for the Freedom EVO Workstation This section lists the instrumentation and labware requirements for the Identity Automation DNA IQ System method on the Freedom EVO Workstation. 5.A. Instrumentation Requirements for the Identity Automation DNA IQ System Method on the Freedom EVO Workstation Minimum Installation Requirements The following is a list of Tecan parts and their corresponding part numbers required for the Identity Automation DNA IQ System method on a Freedom EVO Workstation. Revised 3/12 Page 3

4 Tecan Description Quantity Part Number Tecan Freedom EVO 100 instrument (or larger) 1 Contact Tecan configured with 500µl syringes, 4- or 8-tip Disposable Tip LiHa Arm and RoMa Gripper (Freedom EVOware Standard software) Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier DiTi Carrier, 4-Position, for re-racking DiTi Tips Shelf, 16-Position (4 sites with 4 shelves/site) Trough/DiTi Carrier, 100ml, 3-Position, with 3 Dedicated Positions for reusing Disposable Tips Tube Carrier, 1.5ml or 2.0ml Microcentrifuge with Hinged Cap, 16-Position Te-Shake Microplate Shaker Unit Microplate Nest, 1-Position, with Hold Down, Te-Shake Mounting Plate, Te-Shake 1-Position with 2 additional Microplate Positions EchoTherm Dry Bath, Torrey Pines Scientific, IC20, Electronic Chilling/Heating Microplate Adaptor, Torrey Pines Scientific, IC20/IC22/IC Mounting Plate, Torrey Pines Scientific EchoTherm Dry Bath, IC20/IC22/IC ,000µl LiHa Disposable Tips with Filter 1 per run and µl LiHa Disposable Tips with Filter 2 per run and Trough, Disposable, 100ml, Polypropylene Natural 3 per run or Trough, Disposable, 100ml, Polypropylene Gray These parts are included in the Tecan Package # Uses less than half of a box of tips. Full Work-Flow Requirements The following is a list of Tecan parts and their corresponding part numbers 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 100 or 150 worktable is required for full work-flow automation; please contact your local sales representative for more information. Tecan Part Description Quantity Number Tecan Freedom EVO 100 instrument (or larger) 1 Contact configured with 4 or 8 500µl syringes and Tecan Disposable Tip LiHa Arm (Freedom EVOware Standard software) Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier DiTi Carrier, 4-Position, for re-racking DiTi Tips Shelf, 16-Position (4 sites with 4 shelves/site) Microplate Carrier, 3-Position, Landscape (MP 3Pos) Trough/DiTi Carrier, 100ml, 3-Position, with 3 Dedicated Positions for reusing Disposable Tips Tube Carrier, 1.5ml or 2.0ml Microcentrifuge with Hinged Cap, 16-Position Page 4 Revised 3/12

5 Full Work-Flow Requirements (continued) Tecan Part Description Quantity Number Te-Shake Microplate Shaker Unit Microplate Nest, 1-Position, with Hold Down, Te-Shake Mounting Plate, Te-Shake 1-Position with 2 additional Microplate Positions EchoTherm Dry Bath, Torrey Pines Scientific, IC20, Electronic Chilling/Heating Microplate Adaptor, Torrey Pines Scientific, IC20/IC22/IC Mounting Plate, Torrey Pines Scientific EchoTherm Dry Bath, IC20/IC22/IC ,000µl LiHa Disposable Tips with Filter and µl LiHa Disposable Tips with Filter and µl LiHa Disposable Tips with Filter Trough, Disposable, 100ml, Polypropylene Natural or Trough, Disposable, 100ml, Polypropylene Gray These parts are included in the Tecan Package # Uses less than half of a box of tips. 5.B. Labware Requirements for the Identity Automation DNA IQ System Method on the Freedom EVO Workstation The following is a list of Promega parts and their corresponding part numbers required for the Identity Automation DNA IQ System method on a Freedom EVO Workstation. Promega Part Description Quantity Promega Cat.# MagnaBot 96 Magnetic Separation Device 1 V8151 1/4 inch Foam Spacer 1 Z3301 Deep Well Heat Transfer Block 1 V ml, Square-Well Deep Well Plate or 1 per run V Deep Well Plate from the Slicprep 96 Device (for preprocessed solid samples) 1 per run V1391 Pyramid-Bottom Reservoir, 12-Column 1 per run V ml, Round-Bottom Deep Well Plate 4 per run1 V well PCR plate or strip tubes on plate stand 1 per run (user-selected) 1Two of the four plates are used under the 200µl tips to collect droplets; two are used as processing plates in the method. Revised 3/12 Page 5

6 5.C. Freedom EVO Workstation Initial Deck Configuration MA Page 6 Figure 1. Freedom EVO Workstation initial deck configuration. For more details, see Figures 3 6. Grid 1 Trough/DiTi Carrier, 100ml, 3-Position, with associated tip rack positions Position 1 (rear) 100ml trough Position 2 (middle) 100ml trough Position 3 (front) 100ml trough Grid 3 DiTi Carrier 4-Position Position 1 (rear) 1,000µl LiHa disposable tips with filter Position 2 (midrear) 200µl LiHa disposable tips with filter1 Position 3 (midfront) 200µl LiHa disposable tips with filter1 Position 4 (front) Empty 1A 1.2ml, Round-Bottom Deep Well Plate is required under the 200µl tip boxes to collect droplets. Grid 5 Shelf, 16-Position Empty (back of deck) Position 4 (upper right) 96-well PCR plate or strip tubes on plate stand (Elution Plate) Position 3 (second Empty 1.2ml, Round-Bottom Deep Well Plate position from right) (Purification Plate 2) Grid 9 Wash Station, LiHa, with DiTi Waste Chute and Trough Carrier Grids Tube Carrier, 1.5ml or 2.0ml Microcentrifuge, with Hinged Cap, 16-Position (if starting samples are in tubes) Positions ml microcentrifuge tubes containing samples Turn all open caps so that the caps do not interfere with dispensing steps. Grid 18 Mounting Plate, Te-Shake Position 1 (rear) Empty 1.2ml, Round-Bottom Deep Well Plate (Purification Plate 1) atop MagnaBot 96 Magnetic Separation Device, 1/4 inch Foam Spacer Position 2 (middle) 2.2ml, Square-Well Deep Well Plate or 96 Deep Well Plate from the Slicprep 96 Device containing samples (Sample Plate). Place samples in columns 1 12, depending on the number of samples to be processed. Position 3 (front) Pyramid-Bottom Reservoir, 12-Column Grid 25 Torrey Pines EchoTherm Dry Bath, Deep Well Heat Transfer Block Revised 3/12

7 5.D. Freedom EVO Workstation Variable Inputs The DNA IQ Graphical User Interface (GUI; Figure 2) prompts you to enter userdefined variables at the start of the automated method. These variables are defined by the user based on processing needs. Input Variables Starting Sample Vessel (Plate or Tubes) Sample Type (Aqueous, Lysis or Differex ) Sample Volume (0 400µl) Elution Volume (40 100µl) The Elution Volume variable corresponds to the final volume of Elution Buffer added to each sample well (i.e., µl). If a volume outside of this range is entered, the default volume of 40µl or 100µl will be used. Note that the recovered elution volume in the Elution Plate at the end of the method will be less than the volume of Elution Buffer added due to evaporation on the heater. The average loss is ~5 10µl. Number of Samples to Process (1 96) How Many 1ml Tips Have Been Used? How Many Sample/Elution 200µl Tips Have Been Used? 9216TA Figure 2. DNA IQ System on the Freedom EVO Workstation: Variable Input Screen. Revised 3/12 Page 7

8 After all settings have been indicated on the Variable Input Screen, select "Proceed" to advance to the Deck Setup Information Screen. The DNA IQ GUI will prompt you to add the solutions prepared in Section 4.A to the 100ml troughs as shown in Figure 3. In addition, the volumes of DNA IQ Resin and Lysis Buffer required for the Pyramid- Bottom Reservoir, 12-Column, will be shown (when required). The reagent volumes will vary, depending on the sample number and type. Once you have added the reagents, select "Next Labware". 9217TA Figure 3. DNA IQ System on the Freedom EVO Workstation: Deck Setup Information reagent placement and volumes (Grid 1, 18). Page 8 Revised 3/12

9 The next screen (Figure 4) will prompt you to place tip racks with the appropriate number of tips on the deck. Once the tips are in position, select "Next Labware". 9218TA Figure 4. DNA IQ System on the Freedom EVO Workstation: Deck Setup Information tip rack placement (Grid 3). Revised 3/12 Page 9

10 The next screen (Figure 5) will prompt you to place elution plate or tubes at the top right position of the rail shelf (Position 4) and a clean 1.2ml Round-Bottom Deep Well Plate at Position 3. Verify that a Deep Well Heat Transfer Block is placed on the Torrey Pines heater and that the heater is turned on and set to 85 C. If samples to be processed are in tubes, a prompt indicating placement of the sample tubes will appear. Once these are in place, select "Next Labware". 9219TA Figure 5. DNA IQ System on the Freedom EVO Workstation: Deck Setup Information placement of Elution Plate (Grid 5), clean 1.2ml Round-Bottom Deep Well Plate (Grid 5), and Heat Adaptor (Grid 25). Page 10 Revised 3/12

11 The final screen (Figure 6) will prompt you to place a 1.2ml Round-Bottom Deep Well Plate and 2.2ml Square-Well Deep Well Plate (containing samples if processing samples from a plate) on the deck. When ready to begin the method, select "Proceed with Purification". 9220TA Figure 6. DNA IQ System on the Freedom EVO Workstation: Deck Setup Information processing and sample plate placement (Grids 12 17, 18). 5.E. Freedom EVO Workstation Reagent Dispense Volumes Following the prompts presented in the GUI, dispense the reagents listed below. User-defined input variables enable the automated method to determine the minimum volume of each reagent required in each trough or plate at the start of the method. The following locations require reagents to be dispensed: Grid 1 Trough 1 Dispense Elution Buffer at the volume specified by the GUI (Figure 3). Trough 2 Dispense 1X Wash Buffer at the volume specified by the GUI (Figure 3). Trough 3 Dispense prepared Lysis Buffer at the volume specified by the GUI (Figure 3). Grid 18 Position 3 Dispense DNA IQ Resin Solution at the volume specified by the GUI into the first column of the Pyramid-Bottom Reservoir, 12-Column (Figure 3). Revised 3/12 Page 11

12 6. Description of the Identity Automation DNA IQ System Method This overview describes the general liquid-handling steps required for the Identity Automation DNA IQ System method. 1. Lysate Transfer from Tubes (optional). The liquid-handling robot transfers sample lysates prepared in 1.5ml microcentrifuge tubes to an empty 2.2ml Square-Well Deep Well Plate (Sample Plate). 2. Lysis Buffer Addition. The liquid-handling robot adds prepared Lysis Buffer to each sample in the Sample Plate. 3. DNA IQ Resin Solution Addition. The liquid-handling robot adds DNA IQ Resin Solution to each sample in the Sample Plate. DNA IQ Resin addition is omitted when processing Differex samples; the resin is added during the differential separation. 4. DNA Binding. The Sample Plate is subjected to a series of shaking and incubation steps (10-second shake, 30-second incubation; repeated eight times followed by a final 10-second shake) to allow DNA binding to the DNA IQ Resin. 5. Volume Transfer. The Sample Plate contents are transferred to the first Purification Plate atop the MagnaBot 96 Magnetic Separation Device, which collects the resin at the sides of each well. 6. Sample Lysate Removal. The supernatant (sample lysate) is removed to the Sample Plate, which will now serve as a Lysate Waste Plate. 7. Lysis Buffer Wash. The liquid-handling robot adds prepared Lysis Buffer to each sample well of the Purification Plate. The plate then is moved to the shaker, and the resin is washed by shaking for 30 seconds. 8. Lysis Buffer Wash Removal. The Purification Plate is moved back onto the MagnaBot 96 Magnetic Separation Device, and the supernatant (prepared Lysis Buffer) is removed to the Lysate Waste Plate. 9. 1X Wash Buffer Addition #1. The liquid-handling robot adds 1X Wash Buffer containing alcohols to each sample well of the Purification Plate. The plate is placed on the shaker, and the resin is washed by shaking for 15 seconds. 10. Clean Plate Transfer: The liquid-handling robot transfers the 1X Wash Buffer and DNA IQ Resin to a second Purification Plate on the MagnaBot 96 Magnetic Separation Device X Wash Buffer Removal #1. The supernatant (1X Wash Buffer) is removed from the second Purification Plate and transferred back to the first Purification Plate, which now serves as a 1X Wash Buffer (alcohol) Waste Plate. 12. Washes #2 and #3 with 1X Wash Buffer. The 1X Wash Buffer addition and removal steps are repeated twice for a total of three washes. 13. Heated Drying. The second Purification Plate is moved onto the heater. The system pauses for 5 minutes to allow evaporation of any 1X Wash Buffer in the sample wells. 14. Elution Buffer Addition. The liquid-handling robot adds the desired volume (e.g., 100µl) of Elution Buffer to each sample in the Purification Plate. The Purification Plate is placed on the shaker for 30 seconds to resuspend the resin, then moved to the heater for a 5-minute heated incubation (85 C) to elute DNA from the DNA IQ Resin into the Elution Buffer. Page 12 Revised 3/12

13 15. Elution. After a final 30-second shake, the Purification Plate is moved onto the MagnaBot 96 Magnetic Separation Device, and the supernatant (Elution Buffer containing purified DNA) is removed to the Elution Plate. 16. Method Ends. The Identity Automation DNA IQ System method is now complete. The purified DNA samples in the Elution Plate may be processed immediately or stored at 4 C. 7. Important Considerations 1. Use aerosol-resistant tips to minimize cross-contamination, particularly for casework samples. 2. Thoroughly resuspend the DNA IQ Resin before preparation of the DNA IQ Resin Solution by shaking vigorously. Prior to combining the resin and prepared Lysis Buffer, turn the resin bottle upside-down to ensure that no clumps of resin remain at the bottom of the bottle. 3. Be sure to turn on the heater, and set it to 85 C before running the automated method. 4. The heater set and display temperatures may differ by ~1 C. This is not uncommon for the heater. A difference of ~1 C at the 85 C set temperature will not affect elution efficiency. During heated elution, samples should reach a temperature of approximately 65 C to achieve complete elution. If necessary, adjust temperature or calibration settings for the heater control unit to ensure that proper temperatures are reached. At the appropriate calibration and temperature settings, the surface of the heat block adaptor should be ~70 72 C. 5. The recovered elution volume in the Elution Plate at the end of the method will be less than the volume of Elution Buffer added due to evaporation on the heater and some Elution Buffer lost on the DNA IQ Resin. As much as 5 10µl may be lost. If necessary, adjust the amount of Elution Buffer added to achieve the desired volume recovery. (a)u.s. Pat. Nos. 6,027,945, 6,368,800 and 6,673,631, European Pat. No and Japanese Pat. No , 2012 Promega Corporation. All Rights Reserved. MagnaBot and PowerPlex are registered trademarks of Promega Corporation. Differex, DNA IQ, Identity Automation and Slicprep are trademarks of Promega Corporation. Freedom EVO and Freedom EVOware are registered trademarks of Tecan AG Corporation. Te-Shake is a trademark of Tecan AG Corporation. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. Revised 3/12 Page 13