PrepSEQ Nucleic Acid Extraction Kit

Transcription

1 Quick Reference Card PrepSEQ Nucleic Acid Extraction Kit Note: For safety and biohazard guidelines, refer to the Safety appendix in the PrepSEQ Nucleic Acid Extraction Kit Protocol (PN ). For all chemicals in bold red type, read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Product overview The PrepSEQ Nucleic Acid Extraction Kit prepares high quality microbial DNA and RNA from broth cultures. 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 Storage 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 Overview of pharmaceutical testing 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. For all other samples, use the chemical lysis protocol. After sample lysis using one of these two protocols, proceed with the DNA extraction protocol. 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. Before you begin Before starting your sample extraction: Prepare the following reagents: Binding Solution See Storage on page 1. Wash Buffer See Storage on page 1. 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. Page 2

3 Sample processing workflow for pharmaceutical testing 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 max 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. ; Magnetic particles pellet contains bound DNA Figure 1 Sample processing using the PrepSEQ Nucleic Acid Extraction Kit for pharmaceutical testing Page 3

4 DNA extraction workflow for pharmaceutical testing A DNA extraction workflow is shown below. 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 for pharmaceutical testing Page 4

5 Overview of food testing The PrepSEQ Nucleic Acid Extraction Kit is designed to work with most food types. The kit protocol involves: Enrichment Sample extraction Lysis For the isolation and purification of DNA from overnight broth culture samples, Applied Biosystems recommends a 1-mL sample volume. 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 is recommended. Before you begin Before starting your sample extraction: Prepare the following reagents: Binding Solution See Storage on page 1. Wash Buffer See Storage on page 1. Vortex the Magnetic Particles, then keep at room temperature. Page 5

6 Kit workflow for food testing The figure below shows a sample processing workflow based on the chemical lysis protocol. 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. 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. Step 3: Add 300 µl of Lysis Buffer, then resuspend. 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 3 Food sample preparation using the PrepSEQ Nucleic Acid Extraction Kit with the chemical lysis protocol Page 6

7 Kit workflow for food testing The figure below shows a sample processing workflow based on the Proteinase K lysis protocol. 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 4 Food sample preparation using the PrepSEQ Nucleic Acid Extraction Kit with the Proteinase K lysis protocol Page 7

8 Notes Copyright 2009, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: PLEASE REFER TO THE PREPSEQ NUCLEIC ACID EXTRACTION KIT PRODUCT INSERT AND PROTOCOL FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Applied Biosystems and AB (Design) are registered trademarks, PrepSEQ is a trademark of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. 01/ Part Number Rev. B