DNA Isolation Kit. Catalog Number D USER GUIDE. Publication number MAN Rev. 06

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1 USER GUIDE DNA Isolation Kit Catalog Number D Publication number MAN Rev. 06 For Research Use Only. Not for use in diagnostic procedures.

2 Information in this document is subject to change without notice. Limited Use Label License: Research Use Only The purchase of this product conveys to the purchaser the limited, non transferable right to use the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com. Disclaimers LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) 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. Professional Use Only These products are for professional use only. Trademarks The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. KimWipes is a registered trademark of Kimberly Clark Corporation. 2012, Life Technologies Corporation. All rights reserved. 2 DNA Isolation Kit

3 Contents Product Overview... 4 Product description... 4 Kit usage... 4 DNA Isolation Kit contents and storage... 5 General purpose supplies required but not included... 6 Methods... 7 Before starting... 7 Prepare the starting material... 7 Prepare reagents... 7 Isolate DNA... 8 Troubleshooting Appendix A: Limitations and Cautions Precautions Appendix B: Safety Chemical safety Biological hazard safety Documentation and Support Obtain SDSs Obtain support Certificate of Analysis Limited Product Warranty Symbols References DNA Isolation Kit 3

4 Product Overview Product description Product Overview Product description IMPORTANT! Before using this product, read and understand the information in the Safety appendix in this document. Kit usage Use the DNA Isolation kit to isolate DNA from whole blood and buffy coat samples for PCR and other laboratory procedures. To use the DNA Isolation Kit, you will: 1. Lyse red blood cells and treat the remaining white blood cells with detergent and proteases to release DNA from the nuclei. 2. Use a protein clearing agent to precipitate proteins and RNA. 3. Precipitate DNA with ethanol. 4. Resuspend samples in water or DNA Suspension Buffer (e.g., TE Buffer) for use in various protocols. The processing time is approximately minutes, depending on the number of samples. You may measure the DNA by UV spectrophotometry, fluorimetry or by quantitating on a yield gel against known DNA standards. Ratios of absorbance at 260 nm versus 280 nm (A260/280) are about The yield is 6 14 μg from 0.5 ml of blood. For higher yield, we recommend buffy coat samples. 4 DNA Isolation Kit

5 Product Overview DNA Isolation Kit contents and storage DNA Isolation Kit contents and storage Item Quantity Storage Red Cell Lysis Buffer 2 60 ml 15 C to 30 C room temperature, before use Nuclear Lysis Buffer 7 ml 15 C to 30 C room temperature, before resuspension Protease 28 mg 15 C to 30 C room temperature, before resuspension Protein Clearing Solution 15 ml 15 C to 30 C room temperature DNA Suspension Buffer 15 ml 15 C to 30 C room temperature DNA Isolation Kit 5

6 General purpose supplies required but not included All of the items in this table can be purchased from any major laboratory supplier. Product Information General purpose supplies required but not included Item Description Polypropylene microcentrifuge tubes 2 ml Ice bucket or bench top cooler Microfuge Capable of 12,000 g Pipettors µl Pipette tips Sterile Cotton swabs Vortex mixer Water bath or heat block Set to 65 C 90% Ethanol 70% Ethanol 6 DNA Isolation Kit

7 Methods Before starting Methods Before starting Prepare the starting material or Whole blood Use ml anticoagulated whole blood. Store at room temperature if used within 24 hours from phlebotomy. Refrigerate (0 5 C) for storage up to one week. Freeze whole blood at 20 C for storage up to six months. Buffy coat Use 0.5 ml buffy coat recovered from 1.5 ml fresh whole blood, less than one week from date drawn. Buffy coats cannot be prepared from frozen blood. Note: EDTA or Citrate anticoagulant is preferred. Heparin may inhibit sensitive PCR 4,5. Prepare reagents Nuclear Lysis Buffer Store at room temperature. Clouding white precipitates may appear if the solution is cooled below room temperature. Warm to room temperature and mix thoroughly before using. Use to dilute Protease. Protease (Pronase E) Store at room temperature until ready to use. 1. Add the entire contents of the Nuclear Lysis Buffer to the Protease. Mix gently until resuspended. 2. Aliquot in convenient volumes (125 μl for one sample) and store at 20 C for up to 12 months. Note: After thawing, store the solution at 4 C for up to 2 weeks. Red Cell Lysis Buffer Chill on ice or at 0 5ºC before use. 90% Ethanol Chill on ice or at 0 5ºC before use. DNA Isolation Kit 7

8 Methods Isolate DNA Isolate DNA 1. Add 500 μl of whole blood or buffy coat to a 2.0 ml polypropylene microcentrifuge tube. Note: For increased yields, increase the volume of whole blood up to 1.5 ml. Use the same amount of kit reagents. To accommodate the final total volume of blood and red cell lysis buffer in the 2.0 ml tube, prepare buffy coat or split the total volume of whole blood and red cell lysis buffer (2.5 ml) into two 2.0 ml polypropylene tubes. 2. Add 1 ml of cold (0 5 C) Red Cell Lysis Buffer to the 500 μl of whole blood or buffy coat. Vortex for 30 seconds. 3. Centrifuge the tube for 1 minute at 10,000 12,000 g. 4. Carefully decant the hemolyzed supernatant. Blot the rim of the tube on a clean lint free disposable laboratory wipe (e.g., Kim Wipe ). Note: A white to red pellet will be present at the bottom of the tube. 5. Add 1 ml cold (0 5 C) Red Cell Lysis Buffer. Vortex the tube for 5 seconds. 6. Centrifuge for 1 minute at 10,000 12,000 g. 7. Carefully decant the supernatant and blot the rim of the tube (see step 4). 8. Add 125 μl of Protease in Nuclear Lysis Buffer (4 mg/ml) to the pellet. 9. Vortex for 5 seconds. Note: The pellet may not be in complete resuspension at this point. 10. Place the sample in a 65 C heat block or water bath for 10 minutes. Vortex every 2 3 minutes. Note: The crude lysate can be stored at 4 C overnight or at 20 C for up to one week. 11. Add 275 μl of the Protein Clearing Solution to the sample. 12. Vortex for 5 seconds. 13. Incubate the tube for 10 minutes at 0 5 C (on ice, in bench top cooler, or in refrigerator). 14. Centrifuge the tube for 5 minutes at > 12,000 g. 15. Carefully transfer the supernatant (containing the DNA) to a clean, labeled, 2.0 ml tube. Do not disturb the protein and cell debris pellet at the bottom of the tube. Pipet carefully; the solution may be viscous. 16. Slowly add 500 μl of ice cold (0 5 C) 90% ethanol. 17. Precipitate the DNA by inverting the tube 6 10 times. The DNA will appear as a stringy, white to translucent, mass. If no DNA is visible, place the tube in a freezer ( 20 C) for 10 minutes. 18. Centrifuge the DNA pellet at >12,000 g for 5 minutes. 19. Carefully remove the supernatant by decanting or aspirating using a 1 ml pipettor, being careful not to disrupt the pellet. 8 DNA Isolation Kit

9 Methods Isolate DNA 20. Add 1 ml 70% ethanol, vortex 5 seconds, and centrifuge again for 1 2 minutes at >12,000 g. 21. Carefully remove the ethanol by decanting or by aspirating with a 1 ml pipettor. Air dry the DNA pellet by inverting the tube with the cap open over a clean lintfree disposable laboratory wipe (e.g., Kim Wipe ) for 2 3 minutes to evaporate the ethanol. Use a cotton swab to adsorb any residual ethanol from sides and top of tube, avoiding the DNA pellet. 22. Add 150 μl of DNA Suspension Buffer or sterile distilled water to the tube. 23. Incubate the tube for 15 minutes at 65 C in a water bath or heat block to dissolve the DNA sample. 24. Vortex gently to resuspend. Test the DNA for complete dissolution by pipetting up and down a few times. If clumps of undissolved DNA are present, return the tube to 65 C until you observe complete dissolution. DNA Isolation Kit 9

10 Methods Troubleshooting Troubleshooting Observation Possible Causes Recommended Action Flocculence of kit reagents (The Nuclear Lysis Buffer appears cloudy with a white flocculent precipitate) Inhibition of downstream enzymatic reactions Storing the Nuclear Lysis Buffer at low temperatures Magnesium sensitive applications inhibited by chelation with EDTA Bring the Nuclear Lysis Buffer to room temperature and mix thoroughly before adding to sample. The DNA Suspension Buffer contains Tris buffer (10 mm) and EDTA (1 mm). Adjust the Mg 2+ concentration for magnesium sensitive applications. Low DNA Yield Over dried DNA pellet Be careful not to over dry the DNA pellet as it then becomes difficult to dissolve. Degraded DNA Unbuffered DNA, in storage, is subject to degradation resulting from falling ph as well as from free/random nuclease Store DNA, concentrated (>1 µg/µl), in DNA Suspension Buffer. 10 DNA Isolation Kit

11 Appendix A: Limitations and Cautions Precautions Appendix A: Limitations and Cautions Precautions Blood samples older than one week may produce poor yields and/or poor quality DNA. The yield is dependent on the white cell count of the sample. For samples with known low counts (<3000 WBC/Mm 3 ), centrifuge whole blood (< 2000 g for 15 minutes) to obtain the buffy coat and follow the procedure for whole blood. Heparin has been shown to inhibit some PCR methods 4,5. Use polypropylene tubes and tips for DNA isolation. DNA may adhere to products of other plastic manufacture. Observe universal safety precautions when handling blood samples. Use caution when vortexing samples. Over vortexing or rough handling can result in sheared or degraded DNA DNA Isolation Kit 11

12 Appendix B: Safety General Safety Appendix B: Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the Documentation and Support section in this document. All testing should be performed in accordance with local, regional and national acceptable laboratory accreditation standards and/or regulations. 12 DNA Isolation Kit

13 Appendix B: Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the Documentation and Support section in this document. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturerʹs cleanup procedures as recommended in the SDS. Handle chemical wastes in a fume hood. Ensure use of 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.) After emptying a waste container, seal it with the cap provided. Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure that the 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. DNA Isolation Kit 13

14 Appendix B: Safety Biological hazard safety Biological hazard safety 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: In the U.S.: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at: Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ), found at: 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: In the EU: Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization (WHO) Laboratory Biosafety Manual, third edition, found at: WHO_CDS_CSR_LYO_2004_11/en/ 14 DNA Isolation Kit

15 Documentation and Support Obtain SDSs Obtain support Certificate of Analysis Limited Product Warranty Safety Data Sheets (SDSs) are available from For the SDSs of chemicals not distributed by Life Technologies, contact the chemical manufacturer. For the latest services and support information for all locations, go to: At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support (HLATechSupport@lifetech.com) Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are provided with the product or are available upon request. Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at DNA Isolation Kit 15

16 Symbols Symbol Description Manufacturer Number of Tests Consult Instructions for Use Temperature Limitation (range) Lower Temperature Limitation Upper Temperature Limitation Use By Catalog Number Batch Code Read SDS Read Safety Data Sheet Keep Away from Sunlight Warning: Product may contain biohazardous material Serial Number Date of Manufacture Authorized Representative in the European Community Warning: Attention, see instructions for use Caution, Risk of Electric Shock 16 DNA Isolation Kit

17 References 1. S.A Miller, et al. "A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Research, 1988, v 16 #3, p Current Protocols in Molecular Biology, 1990 Supplement Sambrook, et al. Molecular Cloning. A Lab Manual 1990, Cold Spring Harbor Laboratory, Section E E. Beutler, Interference of Heparin with the Polymerase Chain Reaction. Biotechniques, 1990m v 9 #2, p Effect of Heparin on Polymerase Chain Reaction. Lancet, June 11, 1994, v 343, DNA Isolation Kit 17

18 12 February 2013

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