PrepFiler Automated Forensic DNA Extraction Kit

Transcription

1 QUICK REFERENCE PrepFiler Automated Forensic DNA Extraction Kit Publication Part Number Revision Date 23 November 2011 (Rev. A) Note: For safety and biohazard guidelines, refer to the Safety section in the PrepFiler Automated Forensic DNA Extraction Kit User Guide (Part no ). For every chemical, read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Prepare the reagents and samples Perform lysis: 1.5-mL standard protocol Perform lysis: 96-well plate standard protocol Set up the automation instrument Run automated DNA extraction Kit contents and storage conditions Workflow for manual lysis and automated extraction Prepare the reagents and samples (see page 1 and page 2) Determine sample size or input amount Prepare reagents Select one lysis protocol per run Perform lysis and substrate removal Set up the automation instrument (see page 3) (HID EVOlution Combination System only) Set up the carriers and labware for extraction Perform routine maintenance Place DiTis on the worktable Set up reagents on the workstation Set up lysate, processing, and elution plates and/or Run automated DNA extraction (see page 5) Set up sample information Select and run a script Complete the run Prepare the reagents and samples Determine sample size or input amount Example sample Example sample input Liquid samples (blood, saliva) Up to 40 µl Blood (on FTA paper or fabric) Body fluids (saliva, semen) on fabric Body fluids on swabs (buccal and other body fluids) Prepare reagents Up to 25-mm 2 (cutting or punch) Up to 25-mm 2 (cutting or punch) Up to one swab It is not necessary to use an entire sample punch or swab. 1. Before each use, incubate the Magnetic Particles at 37 C for 10 minutes, then vortex at medium speed until the particles are completely resuspended. 2. If the PrepFiler Lysis Buffer contains precipitate, heat the buffer solution to 37 C for 15 minutes, then vortex the bottle for 5 seconds. 3. Prepare Wash Buffers A and B before first use: a. Mix 260 ml of PrepFiler Wash Buffer A Concentrate with 740 ml of freshly-opened 95% ethanol in a separate, clean container to prepare a 1 solution. b. Mix 200 ml of PrepFiler Wash Buffer B Concentrate with 300 ml of freshly-opened 95% ethanol in a separate, clean container to prepare a 1 solution. 4. Thaw or prepare a fresh 1.0 M solution of DL-dithiothreitol (DTT) in molecular-biology grade DNase-free water. Select one lysis protocol per run If your sample contains no substrate, or includes a substrate that can be submerged using 300 µl of lysis buffer, use the: 1.5-mL standard protocol (page 2) or 96-well plate standard protocol (page 2) Note: With some sample types and inputs, the tube lysis protocol may be the preferred lysis method. Refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for more information. For liquid samples, or samples that include a substrate that requires more than 300 µl of lysis buffer to submerge your sample, refer to the PrepFiler Automated Forensic DNA Extraction Kit User View the output file and the report

2 Perform lysis: 1.5-mL standard protocol Perform lysis 1. Bring the shaking incubator to 70 C. 2. Place each sample in a PrepFiler Spin tube or 1.5-mL microcentrifuge tube. 3. Immediately before each experiment, combine the lysis buffer solution components, then gently mix. Lysis buffer solution component One reaction 4. Add 300 µl of the lysis buffer solution to each sample. If the lysis buffer does not cover the sample substrate, refer to the PrepFiler Automated Forensic DNA Extraction Kit User 5. Cap the, vortex the for 5 seconds, then centrifuge briefly. 6. Place the in a thermal shaker and incubate at 900 rpm and 70 C for 60 minutes. Remove substrate (if present) from sample lysate 1. Label up to 96 new. 2. Centrifuge the sample for 2 seconds. 3. Insert a PrepFiler Filter Column into a new 1.5-mL PrepFiler Spin Tube, then carefully transfer the sample tube contents into the filter column. 4. Cap the filter column/spin tube, then centrifuge for 2 minutes at 13,000 to 16,000 g. 96 reactions PrepFiler Lysis Buffer 300 µl 30 ml 1.0 M DTT 3 µl (5 µl for samples containing semen) Recommended volume; includes approximately 4% excess volume to compensate for pipetting losses. 300 µl 5. Check the volume of sample lysate that is collected in the spin tube. If the volume is less than 180 µl, refer to the PrepFiler Automated Forensic DNA Extraction Kit User 6. Remove the filter column from the spin tube, then properly dispose of the filter column. 7. Transfer the lysate into a new, labeled 1.5-microcentrifuge tube. 8. Allow the lysate to come to room temperature, then proceed directly to the automated extraction run. If you cannot proceed directly, the unprocessed lysate is stable for up to 24 hours at room temperature (20 C) in a sealed tube. Do not chill the sample lysate after performing lysis. Perform lysis: 96-well plate standard protocol 1. Bring the shaking incubator to 70 C. 2. Make sure that the Filter/Spin Plate unit is tightly assembled by centrifuging the unit as described in the PrepFiler Automated Forensic DNA Extraction Kit User 3. Label the PrepFiler Spin Plate. 4. Place each sample in a separate well in the top (Filter Plate) of the plate unit. If you are using fewer than 96 samples, refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for correct positioning. 5. Immediately before each experiment, combine the lysis buffer solution components, then gently mix. Lysis buffer solution component One reaction 6. Add 300 µl of the lysis buffer solution to each sample well. If the lysis buffer does not cover the sample substrate, refer to the PrepFiler Automated Forensic DNA Extraction Kit User 7. Immediately seal the plate unit with a new sheet of MicroAmp Clear Adhesive Film. 8. Put the plate unit into a shaking incubator and incubate at 150 rpm and 70 C for 60 minutes. Keep the plate unit horizontal before and during incubation. 9. Place the plate unit in a deep-well centrifuge. 96 reactions PrepFiler Lysis Buffer 300 µl 30 ml 1.0 M DTT 3 µl 300 µl Recommended volume; includes approximately 4% excess volume to compensate for pipetting losses. 10. Fill a deep-well plate with water so that the weight of the deep-well plate is equal to the weight of the assembled Filter/Spin Plate unit, seal the plate, then place the plate in the deep-well centrifuge as a counterweight. 11. Centrifuge the plate unit at 650 g for 1 minute. If all lysate does not pass into the Spin Plate after 1 minute, centrifuge for an additional minute. 12. While holding the bottom plate, separate the Filter Plate (top) from the Spin Plate (bottom) with the MicroAmp Multi-Removal Tool. If the contents of the Spin Plate are shaken during plate separation, place an adhesive cover on the Spin Plate, place the Spin Plate in the deep-well centrifuge, then briefly centrifuge the Spin Plate. 13. Properly dispose of the (top) Filter Plate, which contains the sample substrate, then proceed directly to the automated extraction run. If you cannot proceed directly, the unprocessed lysate is stable for up to 24 hours at room temperature (20 C) in a sealed Spin Plate. Do not chill the sample lysate after performing lysis. 2 PrepFiler Automated Forensic DNA Extraction Kit Quick Reference Guide

3 Set up the automation instrument 1. (HID EVOlution Combination System only) If the HID EVOlution Combination System was last run for qpcr/ STR setup, refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for instructions on setting up the extraction-specific carriers and labware. 2. Perform routine maintenance as described in the PrepFiler Automated Forensic DNA Extraction Kit User Guide, and run the appropriate maintenance script(s): Before starting the run, if... It is the first run of the day It is not the first run of the day When you run DailyStartUp or Flush, you see: Air bubbles in the lines and/or Intermittent flow from a DiTi cone There are one or more DiTis on the liquid handling arm (LiHa)...then run the script PrepFiler_DailyStartUp or Combo_DailyStartUp PrepFiler_Flush or Combo_Flush PrepFiler_Flush or Combo_Flush one or more times until: There are no visible air bubbles and Flow from the DiTi cones is constant PrepFiler_Drop_DiTis or Combo_Drop_DiTis 3. Place DiTis on the worktable: Three full trays of 1000-µL DiTis into the DiTi racks on the rear shelf (shelf positions 5, 6, and 7). Three full trays of 1000-µL DiTis into the DiTi racks on grid 35, positions 1 through 3. Three full trays of 200-µL DiTis into the carrier on grid 29, positions 1 through Prepare the reagents according to Prepare reagents on page Place the troughs on the worktable according to Table 2 and Table 3 on page Add the reagent volumes specified in Table 1 to the appropriate trough. 7. Gently invert two of PrepFiler Magnetic Particles, briefly centrifuge the at low speed, then open the. Place the two of Magnetic Particles on the worktable in the first two slots of the metal rack on grid 13, position Place the PrepFiler Processing Plate on the Te-Shake adapter with well A1 in the top left position (grid 19, position 3). 9. Set up the eluate or plates. If you want DNA eluate to be collected: In a plate Place a MicroAmp Optical 96-Well Reaction Plate with well A1 in the top left position (grid 13, position 1). In 1.5-mL Place the first empty 1.5-mL tube in the tube racks in rack S1, position 1, then continue placing empty from back to front in vertical columns. Open each tube and secure the tube caps in a fixed upright position. 10. Set up the lysate. If the lysate is in: 1.5-mL Place the first sample tube in the tube racks, then continue placing sample from back to front in vertical columns. Open each tube and secure the tube caps in a fixed upright position. A PrepFiler Spin Plate Place the Spin Plate with well A1 in the top left position (grid 13, position 3). Table 1 Reagent volumes. Using the volumes shown in the table below, calculate the PrepFiler reagent volumes you need. Isopropanol Reagent Protocol Standard (300-µL) lysis protocols Large-sample (500-µL) lysis protocols Reactions (DNA samples) in run Required reagent volume per reaction Required overfill volume per run Required dead volume per run A B C D Minimum required volume for 96 samples (including overfill and dead volume) (A B)+(A B C)+D Up to µl 15% 5 ml 25 ml Up to µl 15% 5 ml 40 ml Prepared Wash Buffer A Up to µl 15% 5 ml 105 ml Prepared Wash Buffer B Up to µl 15% 5 ml 40 ml Elution Buffer Up to µl 15% 5 ml 11 ml Overfill (excess volume) is necessary to compensate for evaporation and pipetting losses during the run. An extra 5 ml per trough is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid, not air, enters the tips. For example, the required volume of isopropanol for 96 samples when using the standard lysis protocol is ( µl) + ( µl 0.15) + 5 ml = ml ml + 5 ml = ml, rounded up to 25 ml. The large-sample (500-µL) protocols were not tested as part of the validation studies that were performed by Life Technologies. If you intend to use the large-sample protocols, perform the appropriate validation studies. PrepFiler Automated Forensic DNA Extraction Kit Quick Reference Guide 3

4 Reference: Worktable layouts Refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for plate-to- and plate-to-plates worktable layouts. Table 2 Tubes-to- workstation layout 1 2 Site number Site number Site number S1 S2 S3 S4 S5 S6 L1 L2 L3 L4 L5 L6 11 Grid numbers Elution tube racks S1 to S6 with microcentrifuge 2. Lysate tube racks L1 to L6 with microcentrifuge 3. Block for PrepFiler Magnetic Particles 4. Magnetic Ring Stand 5. PrepFiler Processing Plate on Te-Shake adapter 6. Isopropanol trough 7. Lysate waste trough 8. Elution Buffer trough 9. Wash Buffer B trough 10. Wash Buffer A trough 11. DiTi waste unit µL disposable pipette tips (DiTis) µL DiTis Rear shelf with 1000-µL DiTis in shelf positions 5, 6, and 7 (not shown) Table 3 Tubes-to-plate workstation layout 1 Site number Site number Site number S1 S2 S3 S4 S5 S6 L1 L2 L3 L4 L5 L6 11 Grid numbers Lysate tube racks L1 to L6 with microcentrifuge Well Elution Plate 3. Block for PrepFiler Magnetic Particles 4. Magnetic Ring Stand 5. PrepFiler Processing Plate on Te-Shake adapter 6. Isopropanol trough 7. Lysate waste trough 8. Elution Buffer trough 9. Wash Buffer B trough 10. Wash Buffer A trough 11. DiTi waste unit µL disposable pipette tips (DiTis) µL DiTis Rear shelf with 1000-µL DiTis in shelf positions 5, 6, and 7 (not shown) 4 PrepFiler Automated Forensic DNA Extraction Kit Quick Reference Guide

5 Run automated DNA extraction Set up sample information Refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for options for entering sample information to the HID EVOlution Extraction System software. Select and run a script 1. On your desktop, click to start the EVOware Standard software, then enter your user name and password. 2. Select Run an existing script, then click. 3. In the Selection dialog box, select the appropriate script for your system, plate/tube selections, and lysis protocol, then click. If you used this lysis protocol... And you want the eluted DNA in... And you are using the HID EVOlution system... HID EVOlution Extraction System HID EVOlution Combination System 1.5-mL standard protocol (see page 2) 96-well plate standard protocol (see page 2) 1.5-mL large-sample protocol (refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide) 96-well plate large-sample protocol (refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide) PrepFiler V1_SP2 PrepFiler Combo_V1_SP1 a 96-well plate PrepFiler plate_v1_sp2 PrepFiler platecombo_v1_sp1 PrepFiler_plate V1_SP2 PrepFiler_plate_Combo_V1_SP1 a 96-well plate PrepFiler_plate_plate_V1_SP2 PrepFiler_plate_plateCombo_V1_SP1 PrepFiler 500_V1_SP2 PrepFiler 500Combo_V1_S P1 a 96-well plate PrepFiler plate500_v1_sp2 PrepFiler plate500combo_v1_s P1 PrepFiler_plate_500_V1_SP2 PrepFiler_plate_500Combo_V1_S P1 a 96-well plate PrepFiler_plate_plate500_V1_SP2 PrepFiler_plate_plate500Combo_V1_SP 1 Version 1 ( V1 ) scripts or later. Contact Life Technologies Technical Support for more information on validated and verified scripts. The large-sample (500-µL) protocols were not tested as part of the validation studies that were performed by Life Technologies. If you intend to use the large-sample protocols, perform the appropriate validation studies. 4. In the Freedom EVOware script dialog, click to run the script, then click in the EVOware Runtime Controller. The Freedom EVOware runtime controller opens, the system initializes, and the liquid-handling arm (LiHa) and Robotic Manipulator arm (RoMa) move. 5. Refer to the PrepFiler Automated Forensic DNA Extraction Kit User Guide for more information about running the script, completing the run, and viewing the output file and report. PrepFiler Automated Forensic DNA Extraction Kit Quick Reference Guide 5

6 Kit contents and storage conditions Each PrepFiler Automated Forensic DNA Extraction kit contains materials sufficient for up to 960 extractions when used with the applicable standard lysis protocol. Plastics are sold separately. Store all kit components at room temperature (18 C to 25 C). Table 4 Materials in PrepFiler Automated Forensic DNA Extraction Kit (Part no ) Reagent PrepFiler Lysis Buffer PrepFiler Magnetic Particles PrepFiler Wash Buffer A Concentrate PrepFiler Wash Buffer B Concentrate PrepFiler Elution Buffer Description One bottle, 500 ml 13, 1.5 ml each One bottle, 500 ml One bottle, 250 ml One bottle, 200 ml Table 5 Plastics (sold separately) Part Number Reagent PrepFiler 96-Well Spin Plate and Filter Plate PrepFiler 96-Well Processing Plate PrepFiler Spin Tubes and Filter Columns AM12450 Non-stick RNase-free Microfuge Tubes (1.5-mL), certified DNaseand RNase-free Description 10 sets 10 plates 300 Spin Tubes and 100 Filter Columns 250 For Research, Forensic, or Paternity Use Only. Not intended for any animal or human therapeutic or diagnostic use. NOTICE TO PURCHASER: PLEASE REFER TO THE PREPFILER AUTOMATED FORENSIC DNA EXTRACTION KIT PRODUCT INSERT AND USER GUIDE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION Life Technologies Corporation. All rights reserved. of Life Technologies Corporation or their respective owners. Freedom EVO, EVOware, Te-Shake, and HID EVOlution are trademarks of Tecan Group AG. Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Toll Free in USA For support visit