Protocol. PrepSEQ Nucleic Acid Extraction Kit

Transcription

1 Protocol PrepSEQ Nucleic Acid Extraction Kit

2 Copyright 2009, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, IN- CLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. NOTICE TO PURCHASER: LIMITED LICENSE The purchase price of this product includes a limited, non-transferable immunity from suit under U.S. patent claims and corresponding patent claims outside the United States for using only this amount of product in the practice of the 5 nuclease process solely in environmental testing, quality control/quality assurance testing, food and agricultural testing, including reporting results of purchaser s activities in environmental testing, quality control/quality assurance testing, food and agricultural testing for a fee or other commercial consideration and also for the purchaser s own internal research and development activities. No right under any other patent claims (such as apparatus or system claims) and no right to perform the 5 nuclease process for any other purpose, is hereby granted expressly, by implication, or by estoppel. This product is for environmental testing, quality control/ quality assurance testing, food and agricultural testing and research purposes only. Diagnostic uses require a separate license from Roche. Further information regarding 5 nuclease licensing program and commercial service licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: Applied Biosystems and AB (Design) are registered trademarks and PrepSEQ is a trademark of Applied Biosystems, Inc. or its subsidiaries in the US and/or certain other countries. All other trademarks are the sole property of their respective owners. Part Number Rev. C 06/2010

3 Contents Preface v Safety information v How to use this guide vi How to obtain support vi Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Product overview Required materials Preparation and extraction of lysate DNA Troubleshooting Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Product overview Required materials Preparation and extraction of lysate DNA Troubleshooting Appendix A Safety Chemical safety Chemical waste safety Biological hazard safety Chemical alerts Documentation Related documentation iii

4 Contents iv

5 Preface Preface Safety information Note: For general safety information, see this Preface and Appendix A, Safety on page 25. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the Safety Appendix for the complete alert on the chemical or instrument. Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below: IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. MSDSs The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining MSDSs, see Appendix A. IMPORTANT! For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer. v

6 Preface How to use this guide How to use this guide Text conventions This guide uses the following conventions: Bold text indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow symbol ( ) separates successive commands you select from a drop-down or shortcut menu. For example: Select File Open Spot Set. Right-click the sample row, then select View Filter View All Runs. User attention words Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. How to obtain support For the latest services and support information for all locations, go to At the Applied Biosystems web site, you can: Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches vi

7 Product overview Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing The PrepSEQ Nucleic Acid Extraction Kit provides a simple way to prepare DNA from broth cultures. PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing Required materials Kit contents The PrepSEQ Nucleic Acid Extraction Kit (PN ) contains reagents for 100 reactions. Kit components are shown in the table below. For information on the kit contents, refer to the Materials supplied section in the packaging insert for your kit. Item Quantity or volume Lysis Buffer, 2 bottles Magnetic Particles, 2 tubes Binding Solution (Isopropanol), 1 empty bottle Wash Buffer Concentrate, 2 bottles Elution Buffer, 1 bottle Proteinase K (PK) Buffer, 1 bottle Proteinase K, 1 tube 50 ml/bottle 1.5 ml/tube NA 26 ml/bottle 25 ml 50 ml 1.25 ml 1

8 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Required materials Storage For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate. WARNING! CHEMICAL HAZARD. Elution Buffer. WARNING! CHEMICAL HAZARD. Proteinase K Buffer. WARNING! CHEMICAL HAZARD. Proteinase K. WARNING! CHEMICAL HAZARD. Magnetic Particles. Room temperature: Lysis Buffer Binding Solution Before its use for the first-time, add 30 ml of 100% isopropanol to the empty Binding Solution bottle. Label the bottle to indicate that isopropanol is added. Wash Buffer Before its use for the first-time, add 74 ml of 95% ethanol to the Wash Buffer Concentrate bottle, mix well, then label the bottle to indicate that ethanol is added. Elution Buffer Proteinase K Buffer Magnetic Particles: 2 to 8 C IMPORTANT! White precipitate occasionally forms in the magnetic particles tube. Extraction experiments show that formation of precipitate does not affect performance. If precipitate forms, incubate the tube at 37 C for 10 minutes, then vortex to completely resuspend the particles. Proteinase K: 20 C For information on storage of kit components, refer to the Storage section in the packaging insert for your kit. 2

9 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Required materials Materials not included in the kit The following table includes materials for using (but not included in) the PrepSEQ Nucleic Acid Extraction Kit. Unless otherwise indicated, many of the listed items are available from major laboratory suppliers (MLS). Equipment, consumables, and reagents Item Equipment Two block heaters, for use with 2-mL tubes, 56 C and 70 C Note: If only one block heater is available, set temperature to 56 C then reset to 70 C after the 56 C incubation. If two block heaters are available, set one to 56 C and the other to 70 C. MLS Source PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing Ice bucket Magnetic stand, 6-tube Refrigerated benchtop microcentrifuge for 1.5- and 2-mL tubes, 2 to 8 C Shaker MLS AM10055 MLS MLS Vortex-Genie 2T Mixer VWR , VWR Vortex Adapter-60, for use with Vortex-Genie AM10014 Consumables Disposable gloves Micropipette tips, aerosol-resistant Pipettors: Positive-displacement Air-displacement Multichannel Pipettes Tubes, conical, 50-mL Microcentrifuge tubes, non-stick RNase-free, 1.5-mL, 1 box (250 tubes/box) Safe-Lock PCR clean microcentrifuge tubes, round-bottom, 2-mL, 1 bag (100 tubes/bag) MLS MLS MLS MLS AM12502 AM12450 VWR Reagents Ethanol, 95% IMPORTANT! Do not use denatured ethanol because it contains components not compatible with the protocol. Isopropanol, 100% MLS MLS 3

10 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Required materials Equipment, consumables, and reagents (continued) Item Source DNase-free, sterile-filtered water MLS 4

11 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA Preparation and extraction of lysate DNA Overview For the isolation of DNA from difficult-to-dissolve, protein-rich tissue samples or samples containing Gram-positive bacteria, Applied Biosystems recommends the Proteinase K lysis protocol described on page 8. For all other samples, use the chemical lysis protocol described on page 7. After sample lysis using one of these two protocols, proceed with the DNA extraction protocol described on page 11. For the isolation and purification of DNA from broth culture samples, Applied Biosystems recommends that the volume of the liquid sample vary from 20 to 100 µl. For liquid samples with a volume >100 µl, centrifuge the liquid sample at g for 5 minutes in an Eppendorf microcentrifuge (to pellet microorganisms), then discard the supernatant. The recommended elution volume is 50 µl. This volume may be increased to 200 µl as necessary. PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing Before you begin Before starting your sample extraction: Prepare the following reagents: Binding Solution Refer to the procedure that is described on page 2. Wash Buffer Refer to the procedure that is described on page 2. Power on the refrigerated centrifuge to allow it to cool down before use. If only one block heater is available, set the temperature to 56 C, then reset to 70 C after the 56 C incubation so that the heater is at the required temperature when needed. If two block heaters are available, set one to 56 C and the other to 70 C. 5

12 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA Sample processing workflow The figure below shows a sample processing workflow. The Proteinase K lysis protocol is recommended for difficult-to-dissolve, protein-rich tissue samples or samples containing Gram-positive bacteria. The chemical lysis protocol is recommended for all other samples. Chemical lysis protocol Step 1: Place the sample in a 1.5 ml-tube. Step 2: Add 300 µl of Lysis Buffer. Vortex for 15 sec. Step 3: Incubate at 70 C for 20 min. Step 4: Vortex for 10 sec at maximum speed. Step 5a: Vortex the magnetic particles for 5 sec. Bind DNA Step 5b: Add 30 µl of Magnetic Particles to the sample mix and vortex for 10 sec at low speed. Step 6: Add 190 µl of Binding Solution. Vortex for 5 sec. Step 7: Incubate with shaking for 7 min. Step 8a: Vortex for 10 sec at low speed. Step 8b: Place the tube in the magnetic stand. Step 8c: Aspirate the supernatant. Proteinase K lysis protocol Step 1: Place the sample in a 1.5 ml-tube. Step 2: Add 200 µl of Proteinase K (PK) Buffer. Mix and resuspend. Step 3: Add 10 µl of Proteinase K (20 mg/ml). Step 4: Incubate at 56 C for 20 min. Step 5: Add 400 µl of Lysis Buffer. Vortex for 15 sec. Note: If necessary you can leave the lysate at room temperature for up to two hours. Step 6: Vortex the sample lysate for 10 sec at maximum speed. Bind DNA Step 7a: Vortex the magnetic particles for 5 sec. Step 7b: Add 30 µl of Magnetic Particles to the sample mix and vortex for 10 sec at low speed. Step 8: Add 400 µl of Binding Solution and vortex for 5 sec. Step 9: Incubate with shaking for 10 min at room temp. Step 10a 10c: Vortex for 10 sec at low speed. Place the tube in the magnetic stand and aspirate. ; Pellet of magnetic particles with bound DNA; Proceed to DNA extraction (page 9) Figure 1 Sample processing using the PrepSEQ Nucleic Acid Extraction Kit 6

13 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA Chemical lysis protocol For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Magnetic Particles. 1. Place the sample in a 1.5 ml microcentrifuge tube. 2. Add 300 µl of the Lysis Buffer to the tube, then vortex the tube for 15 seconds, until the sample is completely resuspended. PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing 3. Incubate the tube at 70 C for 20 minutes. 4. Vortex the tube for 10 seconds at maximum speed. 5. Add the Magnetic Particles to the sample mix: a. Vortex the stock Magnetic Particles for approximately 5 seconds, until resuspension is complete. b. Add 30 µl of the Magnetic Particles to the sample mix, then vortex the tube for 10 seconds at low speed. 6. Add 190 µl of the Binding Solution to the tube, then vortex the tube for 5 seconds. 7. Incubate the mix with shaking (approximately 1000 rpm) at room temperature for 7 minutes. 8. Separate the mix: a. Vortex the tube for 10 seconds at low speed. b. Place tube in the magnetic stand. Note: Complete separation of the magnetic particles pellet containing the bound DNA occurs in 1 to 2 minutes. c. Aspirate the supernatant carefully, then discard as much of the liquid as possible without disturbing the magnetic particles pellet. 9. Proceed to DNA extraction: purification of DNA on page 11. 7

14 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA Proteinase K lysis protocol For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Proteinase K Buffer. WARNING! CHEMICAL HAZARD. Proteinase K. WARNING! CHEMICAL HAZARD. Magnetic Particles. IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-contamination. 1. Place the sample in a 1.5-mL microcentrifuge tube. 2. Add 200 µl of the Proteinase K (PK) Buffer. Mix and resuspend. 3. Add 10 µl of Proteinase K (20 mg/ml) to the sample. 4. Incubate the tube at 56 C for 20 minutes. 5. Add 400 µl of the Lysis Buffer, then vortex the tube for 15 seconds until the sample is completely resuspended. Note: If necessary you can leave the lysate at room temperature for up to 2 hours. 6. Vortex the sample lysate for 10 seconds at maximum speed. 7. Add the Magnetic Particles to the sample mix: a. Vortex the Magnetic Particles for approximately 5 seconds, until resuspension is complete. b. Add 30 µl of the Magnetic Particles to the sample mix, then vortex the tube for 10 seconds at low speed. Note: If a white precipitate forms in the magnetic particles tube, refer to the IMPORTANT paragraph on page Add 400 µl of the Binding Solution to the tube, then vortex the tube for 5 seconds. 9. Incubate with shaking (approximately 1000 rpm) for 10 minutes at room temperature. 8

15 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA 10. Separate the mix: a. Vortex the tube for 10 seconds at low speed. b. Place the tube in the magnetic stand with the magnetic particles pellet oriented toward the magnet. Note: Complete separation of the magnetic particles containing the bound DNA occurs in 1 to 2 minutes. c. Carefully aspirate, then discard as much of the supernatant as possible without disturbing the magnetic particles pellet. PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing 11. Proceed to DNA extraction: purification of DNA on page 11. 9

16 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA DNA extraction workflow The figure below shows a DNA extraction workflow. For details, go to page 11. Wash DNA Step 1: Add 300 µl of Wash Solution. Step 2a 2c: Vortex for 10 sec at setting #7. Place the tubes into a magnetic stand. Aspirate the supernatant without disturbing the magnetic particles pellet. Step 3: Repeat steps 1 and 2 two more times. Step 4: Leave the tube lid open for 5 min to air-dry. Step 1 Add 50 µl of Elution Buffer. Elute DNA Step 2: Vortex for 5 sec at medium speed. Step 3: Incubate at 70 C for 5 min. Step 4: Vortex for 2 sec at medium speed. Step 5: Place the tube into a magnetic stand. Step 6: Transfer the liquid phase to a non-stick tube. Step 7: Store at 20 C. Figure 2 DNA extraction using the PrepSEQ Nucleic Acid Extraction Kit 10

17 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Preparation and extraction of lysate DNA DNA extraction: purification of DNA Wash DNA 1. Add 300 µl of Wash Solution to the tube. 2. Separate the mix: a. Vortex the tube for 10 seconds at speed setting #7 until the pellet is completely resuspended. b. Place the tube in the magnetic stand with the magnetic particles pellet oriented toward the magnet. Note: Complete capture of the magnetic particles containing the bound DNA occurs in 30 seconds. PrepSEQ Nucleic Acid Extraction Kit Protocol for Pharmaceutical Testing c. Carefully aspirate, then discard as much of the supernatant as possible without disturbing the magnetic particles pellet. The pellet contains bound DNA. 3. Repeat step 1 and step 2 two more times. 4. With the tube lid open, air-dry the magnetic particles pellet in the magnetic stand for 5 minutes at room temperature. IMPORTANT! Ethanol in the Wash Solution decreases recovery and causes PCR inhibition. Avoid carrying over ethanol into the elution steps. Elute DNA For the following hazard, see the complete safety alert descriptions in Appendix A on page 25: WARNING! CHEMICAL HAZARD. Elution Buffer. 1. Add 50 µl of Elution Buffer to the tube. Note: The magnetic particles may be difficult to detach from the tube wall. Place the tube in the benchtop microcentrifuge with the magnetic particles pellet oriented toward the center, then spin the tube for 30 seconds to detach the magnetic particles into the Elution Buffer. 2. Vortex the tube for 5 seconds at medium speed. 3. Incubate the tube at 70 C for 5 minutes. 4. Vortex the tube for 2 seconds at medium speed. 5. Place the tube in the magnetic stand for at least 2 minutes (or until magnetic particles pellet is formed). 11

18 Chapter 1 PrepSEQ Nucleic Acid Extraction Kit for Pharmaceutical Testing Troubleshooting 6. Transfer the liquid phase containing the eluted DNA to a non-stick 1.5-mL microcentrifuge tube. Note: The final liquid phase contains extracted DNA. 7. Store the tube at 20 C. Troubleshooting Observation Possible Cause Action Poor extraction efficiency (low yields) Too much starting material Spin speed is too high (pellet too hard) in the preparation step Carryover of ethanol into Elute DNA step 1 on page 11. Magnetic particles are attached too tightly to the tube wall during Elution DNA step 1 on page 11. Magnetic particles are difficult to resuspend during Elute DNA step 1 on page 11. Start with less volume (less material) Pellet using a slower speed Ensure resuspension of the pellet in Lysis Buffer or Proteinase K Buffer before proceeding Thoroughly air-dry the magnetic particles pellet in the magnetic stand (no longer than 15 minutes) at room temperature. Spin the tube for 30 seconds at maximum speed to detach the magnetic particles into the Elution Buffer. Incubate the pellets at 70 C for 7 minutes. Vortex the tubes 3 times during incubation to facilitate resuspension. 12

19 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Product overview Required materials The PrepSEQ Nucleic Acid Extraction Kit prepares high quality microbial DNA and RNA from broth cultures. Kit contents The PrepSEQ Nucleic Acid Extraction Kit for Food Testing (PN ) contains reagents for 100 reactions. Kit components are shown in the table below. For information on the kit contents, refer to the Materials supplied section in the packaging insert for your kit. PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing Item Quantity or volume Lysis Buffer, 2 bottles Magnetic Particles, 2 tubes Binding Solution (Isopropanol), 1 empty bottle Wash Buffer Concentrate, 2 bottles Elution Buffer, 1 bottle Proteinase K (PK) Buffer, 1 bottle Proteinase K, 1 tube 50 ml/bottle 1.5 ml/tube NA 26 ml/bottle 25 ml 50 ml 1.25 ml 13

20 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Required materials Storage For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate. WARNING! CHEMICAL HAZARD. Elution Buffer. WARNING! CHEMICAL HAZARD. Proteinase K Buffer. WARNING! CHEMICAL HAZARD. Proteinase K. WARNING! CHEMICAL HAZARD. Magnetic Particles. Room temperature: Lysis Buffer Binding Solution Before its use for the first-time, add 30 ml of 100% isopropanol to the empty Binding Solution bottle. Label the bottle to indicate that isopropanol is added. Wash Buffer Before its use for the first-time, add 74 ml of 95% ethanol to the Wash Buffer Concentrate bottle, mix well, then label the bottle to indicate that ethanol is added. Elution Buffer Proteinase K Buffer Magnetic Particles: 2 to 8 C IMPORTANT! White precipitate occasionally forms in the magnetic particles tube. Extraction experiments show that formation of precipitate does not affect performance. If precipitate forms, incubate the tube at 37 C for 10 minutes, then vortex to completely resuspend the particles. Proteinase K: 20 C For information on storage of kit components, refer to the Storage section in the packaging insert for your kit. 14

21 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Required materials Materials not included in the kit The following table includes materials for using (but not included in) the PrepSEQ Nucleic Acid Extraction Kit. Unless otherwise indicated, many of the listed items are available from major laboratory suppliers (MLS). Equipment, consumables, and reagents Item Source Equipment Benchtop microcentrifuge (Eppendorf 5415 D or equivalent) MagMAX Express-96 Deep Well Magnetic Particle Processor Stomacher 400 Laboratory Blender Vortexer Disposable gloves Micropipette tips, aerosol-resistant Pipettors: Positive-displacement Air-displacement Multichannel Consumables MLS PN Seward #0400/001/AJ MLS MLS MLS MLS PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing MagMAX Express-96 Deep Well Tip Combs PN MagMAX Express-96 Deep Well Plates PN Microtiter 96-Well Plate VWR # , Thermofisher # Whirl-Pak Filter Bags, 6 9, 24 oz, 250/pkg (Stomacher bags with mesh) Whirl-Pak Filter Bags, 6 9, 24 oz (Stomacher bags without mesh) Microcentrifuge tubes, PCR clean, 1.5-mL VWR # VWR # MLS Reagents Bacterial growth media DNase-free, sterile-filtered water Ethanol, 95% Isopropanol, 100% MLS PN AM9938 MLS MLS microorganism appropriate 15

22 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA Preparation and extraction of lysate DNA Overview The PrepSEQ Nucleic Acid Extraction Kit is designed to work with most food types. The kit protocol involves: Sample enrichment Sample extraction Lysis To isolate DNA from Gram-negative bacteria in food samples, the chemical lysis protocol that is described on page 19 is recommended. To isolate DNA from Gram-positive bacteria in food samples, the Proteinase K lysis protocol that is described on page 21 is recommended. For the isolation and purification of DNA from overnight broth culture samples, a 1-mL sample volume is recommended. The recommended elution volume is 140 µl. If you prepare food samples that yield large pellet samples, such as that found with chocolate, the PrepSEQ preclarification protocol on page 19 is recommended. Before you begin Before starting your sample extraction: Prepare the following reagents: Binding Solution See the procedure that is described on page 14. Wash Buffer See the procedure that is described on page 14. Vortex the Magnetic Particles, then keep them at room temperature. 16

23 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA Kit workflow The figure below shows a sample processing workflow based on the chemical lysis protocol. For details go to page 19. Enrichment Use bacterial growth media appropriate for microorganisms and enrich according to your standard protocols. Chemical lysis protocol Step 1: Transfer 1 ml of sample into a 1.5-mL tube. Step 2: Centrifuge the tube for 3 min at g. Discard the supernatant. Step 3: Add 300 µl of Lysis Buffer, then resuspend. If your sample produces a large food pellet (PrepSEQ preclarification protocol): Transfer 1 ml of sample into a 1.5-mL tube. Centrifuge the tube for 1 min at 4000 g. Collect the supernatant, then transfer to a new 1.5-mL tube. PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing Step 4: Transfer the sample to a microtiter 96-well deep well plate. Step 5a b: Prepare the plates. Step 6: Select DWPrepSEQGN from the MagMAX Express magnetic particle processor, then press Start. Step 7a e: Load the plates, then press Start. Step 8: After 18 min, dispense the Binding Mix: a. Vortex the Magnetic Particles for 5 sec until resuspension is complete. b. Add 30 µl of Magnetic Particles. Swirl the plate. c. Add 180 µl of Binding Solution. Swirl the plate. d. Load the plate into the instrument, then press Start. Step 9: After 30 min, sample preparation is complete. The message Enjoy your DNA is displayed on the screen. Remove the elution plate. Proceed with PCR, or seal the plate and store at 20 C. Figure 1 Food sample preparation using the PrepSEQ Nucleic Acid Extraction Kit with the chemical lysis protocol 17

24 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA Kit workflow The figure below shows a sample processing workflow based on the Proteinase K lysis protocol. For details go to page 21. Enrichment Use bacterial growth media appropriate for microorganisms and enrich according to your standard protocols. Step 1: Transfer 1 ml into a 1.5-mL tube. Proteinase K lysis protocol Step 2: Centrifuge the tube for 3 min at g. Discard the supernatant. Step 3: Add 200 µl of PK Buffer. Mix and resuspend. Step 4: Add 10 µl of Proteinase K (20 mg/ml). Step 5: Transfer the sample to a microtiter 96-well deep well plate. Step 6a b: Prepare the plates. Step 7: Select DWPrepSEQGP from the MagMAX Express magnetic particle processor, then press Start. Step 8a e: Load plates, then press Start. Step 9: After 20 min, dispense the Lysis Buffer: a. Add 300 µl of Lysis Buffer. Swirl the plate. b. Load the plate into the instrument, then press Start. Step 10: After 18 min, dispense the Binding Mix: a. Vortex Magnetic Particles for 5 sec. b. Add 30 µl of Magnetic Particles to each well. Swirl the plate. c. Add 300 µl of Binding Solution to each well. Swirl the plate. d. Load the plate into the instrument, then press Start. Step 11: After 30 min, sample preparation is done. The message Enjoy your DNA is displayed on the screen. Remove the elution plate. Proceed with PCR, or seal the plate and store at 20 C. Figure 2 Food sample preparation using the PrepSEQ Nucleic Acid Extraction Kit with the Proteinase K lysis protocol 18

25 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA Generic enrichment Use bacterial growth media appropriate for microorganisms and enrich according to your standard protocols. Getting started For isolation of DNA from Gram-positive bacteria, proceed to the Chemical lysis protocol below. For isolation of DNA from Gram-negative bacteria, proceed to the Proteinase K lysis protocol on page 21. Chemical lysis protocol For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Elution Buffer. WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate. WARNING! CHEMICAL HAZARD. Magnetic Particles. PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-contamination. 1. Transfer 1 ml of sample into a 1.5-mL microcentrifuge tube. 2. Centrifuge the tube for 3 minutes at g. Note: Discard the supernatant without disturbing the pellet. Remove liquid as quickly as possible to prevent dissipation of pellet. If your sample produces a large pellet at this step, such as found with chocolate food samples, the following PrepSEQ preclarification protocol is recommended: Transfer 1 ml of sample into a 1.5-mL microcentrifuge tube. Centrifuge the tube containing your sample for 1 minute at 4000 g. Transfer supernatant to a new 1.5-mL microcentrifuge tube, without disturbing the pellet. Proceed to step Add 300 µl of the Lysis Buffer to the tube. Resuspend by pipetting up and down, or vortex until the pellet is resuspended. Quick spin for 5 seconds to remove the Lysis Buffer from the tube lid. 4. Transfer the sample to a sterile microtiter 96-well deep well (DW) plate (PN ). 19

26 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA 5. Prepare the plates: a. To prepare the elution plate, add 140 µl of Elution Buffer to those wells of the microtiter 96-well plate that correspond to the microtiter 96-well DW plate containing sample. b. To prepare wash plates 1 and 2, add 300 µl of Wash Buffer to those wells of the microtiter 96-well DW plate by that correspond to the microtiter 96-well DW plate containing sample. 6. Select DWPrepSEQGN from the MagMAX Express magnetic particle processor. Press Start. 7. Load the plates according to the readout. Verify orientation {A1 to A1}. a. Tip combs in microtiter 96-well plate; press Start. b. Elution plate (140 µl of Elution Buffer) In microtiter 96-well plate; press Start. c. Wash plate 2 (300 µl of Wash Buffer) In microtiter 96-well DW plate; press Start. d. Wash plate 1 (300 µl of Wash Buffer) In microtiter 96-well DW plate; press Start. e. Lysis plate (sample in Lysis Buffer) In microtiter 96-well DW plate; press Start. 8. After 18 minutes, the MagMAX Express magnetic particle processor prompts you to dispense the Binding Mix. a. Vortex the Magnetic Particles for 5 seconds until resuspension is complete. b. Add 30 µl of the Magnetic Particles to each well. Swirl the plate. c. Add 180 µl of Binding Solution to each well. Swirl the plate. d. Load the plate back into the instrument. Press Start. 9. When sample preparation is complete, the message Enjoy your DNA is displayed on the screen. Remove the elution plate. Store at 20 C. IMPORTANT! For PCR, add 30 µl of eluate to the lyophilized assay. 20

27 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA Proteinase K lysis protocol For the following hazards, see the complete safety alert descriptions in Appendix A on page 29: DANGER! CHEMICAL HAZARD. Lysis Buffer. WARNING! CHEMICAL HAZARD. Proteinase K Buffer. WARNING! CHEMICAL HAZARD. Proteinase K. WARNING! CHEMICAL HAZARD. Elution Buffer. WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate. WARNING! CHEMICAL HAZARD. Magnetic Particles. IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-contamination. Note: Premix Proteinase K Buffer and Proteinase K prior to dispensing. PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing 1. Transfer 1 ml of sample into a 1.5-mL microcentrifuge tube. 2. Centrifuge the tube for 3 minutes at g. Note: Discard the supernatant without disturbing the pellet. Remove liquid as quickly as possible to prevent dissipation of pellet. 3. Add 200 µl of the Proteinase K (PK) Buffer. Mix, then resuspend. Note: Depending on the number of samples to be run, a master mix may be prepared prior to dispensing; master mix volume = N (200 µl of Proteinase K Buffer + 10 µl of Proteinase K) where N = # of samples. 4. Add 10 µl of Proteinase K (20 mg/ml) to the sample. 5. Transfer the sample to a sterile microtiter 96-well deep well (DW) plate (PN ). 6. Prepare the plates: a. To prepare the elution plate, add 140 µl of Elution Buffer to those wells of the microtiter 96-well plate that correspond to the microtiter 96-well DW plate containing sample. 21

28 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Preparation and extraction of lysate DNA b. To prepare wash plates 1 and 2, add 300 µl of Wash Buffer to those wells of the microtiter 96-well DW plate by that correspond to the microtiter 96-well DW plate containing sample. 7. Select DWPrepSEQGP program from the MagMAX Express magnetic particle processor. Press Start. 8. Load the plates according to the readout. Verify orientation {A1 to A1}. a. Tip combs in microtiter 96-well plate; press Start. b. Elution plate (140 µl Elution Buffer) In microtiter 96-well plate; press Start. c. Wash plate 2 (300 µl of Wash Buffer) In microtiter 96-well DW plate; press Start. d. Wash plate 1 (300 µl of Wash Buffer) In microtiter 96-well DW plate; press Start. e. Lysis plate (sample in PK Buffer) In microtiter 96-well DW plate; press Start. 9. After 20 minutes, the MagMAX Express magnetic particle processor prompts you to dispense the Lysis Buffer. a. Add 300 µl of Lysis Buffer to each well. b. Load the plate into the instrument. Press Start. 10. After 18 minutes, the instrument prompts you to dispense the Binding Mix. a. Vortex the stock Magnetic Particles for 5 seconds until resuspension is complete. b. Add 30 µl of Magnetic Particles to each well. Swirl the plate. c. Add 300 µl of Binding Solution to each well. Swirl the plate. d. Load the plate into the instrument. Press Start. 11. When sample preparation is complete, the message Enjoy your DNA is displayed on the screen. Remove the elution plate. Store at 20 C. IMPORTANT! For PCR, add 30 µl of eluate to the lyophilized assay. 22

29 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Troubleshooting Troubleshooting For food testing Observation Possible cause Action Inhibition of PCR, indicated by nondetection of IPC reaction Magnetic particles were in the elution plate Elution plate contains incompletely removed particulate residue from food sample Removal of sample supernatant before addition of lysis buffer was incomplete Avoid disturbing the magnetic particles during transfer of eluted DNA to the lyophilized assay. Optional: Spin the plate for 30 seconds at 4000 g to pellet the magnetic particles to the bottom of the plate. Or: Place the elution plate on the 96-well magnetic ring stand (PN AM10050) during transfer of sample to the lyophilized assay. Avoid residue during transfer of eluted DNA to lyophilized assay. Optional: Spin the plate for 30 seconds at 4000 g to pellet the food residue to the bottom of the plate. Ensure maximal removal of the supernatant without disturbing the bacterial pellet. PrepSEQ Nucleic Acid Extraction Kit Protocol for Food Testing The bacterial pellet is hard to avoid during removal of supernatant The bacterial pellet is difficult to resuspend The sample was left unattended before aspirating off the supernatant, causing dissipation of the bacterial pellet The size of the bacterial pellet is very small and difficult to see Pellet is too hard The pellet is too large Remove the supernatant immediately following centrifugation. Remove the supernatant carefully, leaving behind up to 50 µl of supernatant, to avoid aspiration of pellet. Ensure maximum resuspension of the pellet in the Lysis Buffer or Proteinase K Buffer before proceeding. Transfer the entire contents, including the incompletely resuspended pellet (if any) to the Lysis Plate. Apply PrepSEQ preclarification protocol. 23

30 Chapter 2 PrepSEQ Nucleic Acid Extraction Kit for Food Testing Troubleshooting 24

31 Appendix A Safety This appendix covers: Chemical safety Chemical waste safety Biological hazard safety Chemical alerts

32 Appendix A Safety Chemical safety Chemical safety Chemical hazard warning WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all relevant precautions. WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument. WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. Chemical safety guidelines To minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About MSDSs on page 27.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended in the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 26

33 Appendix A Safety Chemical waste safety About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs: 1. Go to click Support, then select MSDS. 2. In the Keyword Search field, enter the chemical name, product name, MSDS part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search. 3. Find the document of interest, right-click the document title, then select any of the following: Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you select Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Chemical waste safety Chemical waste hazards CAUTION! HAZARDOUS WASTE. Refer to Material Safety Data Sheets (MSDSs) and local regulations for handling and disposal. WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. 27

34 Appendix A Safety Chemical waste safety Chemical waste safety guidelines To minimize the hazards of chemical waste: Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. Handle chemical wastes in a fume hood. After emptying a waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Waste disposal If potentially hazardous waste is generated when you operate the instrument, you must: Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure the health and safety of all personnel in your laboratory. Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 28

35 Appendix A Safety Biological hazard safety Biological hazard safety General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ; bmbl.od.nih.gov) Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ; nara/cfr/waisidx_01/29cfr1910a_01.html) Your company s/institution s Biosafety Program protocols for working with/handling potentially infectious materials. Additional information about biohazard guidelines is available at: Chemical alerts General alerts for all chemicals Avoid contact with (skin, eyes, and/or clothing). Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Specific chemical alerts WARNING! Dispose of the pipette tips and other contaminated materials in biohazard waste. DANGER! CHEMICAL HAZARD. Lysis Buffer contains guanidine thiocyanate. Exposure causes eye burns and skin irritation. It is harmful if inhaled, absorbed through the skin or if swallowed. Contact with acids or bleach liberates toxic gases. DO NOT ADD acids or bleach to any liquid 29

36 Appendix A Safety Chemical alerts wastes containing DNA Binding Buffer. Avoid breathing vapor. Do not taste or swallow. Use with adequate ventilation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate contains a material that may cause eye, skin, and respiratory tract irritation, and adverse effects on the kidneys and blood and central nervous system. Use with adequate ventilation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING! CHEMICAL HAZARD. Elution Buffer contains a material that may cause eye, skin, and respiratory tract irritation, and adverse effects on the kidneys and blood and central nervous system. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING! CHEMICAL HAZARD. Proteinase K Buffer contains a material that may cause eye, skin, and respiratory tract irritation, and adverse effects on the kidneys and blood and central nervous system. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING! CHEMICAL HAZARD. Proteinase K causes eye, skin, and respiratory tract irritation. Exposure may cause an allergic reaction. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING! CHEMICAL HAZARD. Magnetic Particles cause eye, skin, and respiratory tract irritation. Exposure may cause an allergic reaction. Harmful by inhalation, skin absorption and if swallowed. Do not taste or swallow. Avoid breathing vapor (or dust). Use with adequate ventilation. Avoid contact with eyes and skin. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 30

37 Documentation Documentation Related documentation For additional documentation, see How to obtain support on page vi. For information on new assays and updated product documentation, go to Document title PN PrepSEQ Nucleic Acid Extraction Kit Quick Reference Card : Salmonella spp PrepSEQ Nucleic Acid Extraction Kit Quick Reference Card: Salmonella spp. : Listeria monocytogenes PrepSEQ Nucleic Acid Extraction Kit Quick Reference Card: Listeria monocytogenes PrepSEQ Mycoplasma Nucleic Acid Extraction Kit Protocol PrepSEQ Mycoplasma Nucleic Acid Extraction Kit Quick Reference Card PrepSEQ Rapid Spin Sample Preparation Kit Protocol PrepSEQ Rapid Spin Sample Preparation Kit Quick Reference Card PrepSEQ Rapid Spin Sample Preparation Kit Protocol: Salmonella spp PrepSEQ Rapid Spin Sample Preparation Kit Quick Reference Card: Salmonella spp. PrepSEQ Rapid Spin Sample Preparation Kit Protocol: Listeria monocytogenes PrepSEQ Rapid Spin Sample Preparation Kit Quick Reference Card: Listeria monocytogenes MicroSEQ Listeria monocytogenes Detection Kit Protocol MicroSEQ Listeria monocytogenes Detection Kit Quick Reference Card MicroSEQ Salmonella spp. Detection Kit Protocol MicroSEQ Salmonella spp. Detection Kit Quick Reference Card MicroSEQ Mycoplasma Real-Time PCR Detection Kit Protocol MicroSEQ Mycoplasma Real-Time PCR Detection Kit Quick Reference Card Portable document format (PDF) versions of this guide and the documents listed above are available at Note: To open the documentation available from the Applied Biosystems web site, use the Adobe Acrobat Reader software available at 31