dna isolation kit - thermo fisher...

USER GUIDE

DNA Isolation Kit Catalog Number 761001D

Publication number MAN0002536 Rev. 06 For Research Use Only. Not for use in diagnostic procedures.

2 DNA Isolation Kit

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 [email protected]. 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.

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 ................................................................................................ 10

Appendix A: Limitations and Cautions ............................................ 11

Precautions ......................................................................................................... 11

Appendix B: Safety ....................................................................... 12

Chemical safety .................................................................................................. 13

Biological hazard safety ..................................................................................... 14

Documentation and Support .............................................................................. 15

Obtain SDSs ................................................................................................. 15 Obtain support ............................................................................................. 15 Certificate of Analysis ................................................................................... 15 Limited Product Warranty ............................................................................ 15

Symbols ............................................................................................................. 16

References .......................................................................................................... 17

Product Overview Product description

4 DNA Isolation Kit

Product Overview

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document.

Product description

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 45–60 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 1.7 –1.9. The yield is 6–14 μg from 0.5 mL of blood. For higher yield, we recommend buffy coat samples.

Product Overview DNA Isolation Kit contents and storage

DNA Isolation Kit 5

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

Product Information General purpose supplies required but not included

6 DNA Isolation Kit

General purpose supplies required but not included All of the items in this table can be purchased from any major laboratory supplier.

Item Description

Polypropylene microcentrifuge tubes 2 mL

Ice bucket or bench top cooler —

Microfuge Capable of 12,000 × g

Pipettors 25–1000 µL

Pipette tips Sterile

Cotton swabs —

Vortex mixer —

Water bath or heat block Set to 65°C

90% Ethanol —

70% Ethanol —

Methods Before starting

DNA Isolation Kit 7

Methods

Before starting

Prepare the starting material

Whole blood Use 0.5–1.5 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.

or 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 PCR4,5.

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.

Methods Isolate DNA

8 DNA Isolation Kit

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.

Methods Isolate DNA

DNA Isolation Kit 9

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 lint‐free 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.

Methods Troubleshooting

10 DNA Isolation Kit

Troubleshooting

Observation Possible Causes Recommended Action Flocculence of kit reagents (The Nuclear Lysis Buffer appears cloudy with a white flocculent precipitate)

Storing the Nuclear Lysis Buffer at low temperatures

Bring the Nuclear Lysis Buffer to room temperature and mix thoroughly before adding to sample.

Inhibition of downstream enzymatic reactions

Magnesium sensitive applications inhibited by chelation with EDTA

The DNA Suspension Buffer contains Tris buffer (10 mM) and EDTA (1 mM). Adjust the Mg2+ 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.

Appendix A: Limitations and Cautions Precautions

DNA Isolation Kit 11

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/Mm3), 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 methods4,5.

• 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

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.

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.

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: www.cdc.gov/biosafety

• Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030), found at: www.access.gpo.gov/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: www.cdc.gov

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: www.who.int/csr/resources/publications/biosafety/ WHO_CDS_CSR_LYO_2004_11/en/


Documentation and Support

Obtain SDSs

Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support

For the SDSs of chemicals not distributed by Life Technologies, contact the chemical manufacturer.

Obtain support

For the latest services and support information for all locations, go to:

www.lifetechnologies.com/support

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 ([email protected])

• 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

Certificate of Analysis

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.

Limited Product Warranty

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 www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.


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


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. 1215.

2. Current Protocols in Molecular Biology, 1990 Supplement 9.

3. Sambrook, et al. Molecular Cloning. A Lab Manual 1990, Cold Spring Harbor Laboratory, Section E 10.

4. E. Beutler, Interference of Heparin with the Polymerase Chain Reaction. Biotechniques, 1990m v 9 #2, p. 166.

5. Effect of Heparin on Polymerase Chain Reaction. Lancet, June 11, 1994, v 343, 1509‐10.

12 February 2013

Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288For support visit lifetechnologies.com/support or email [email protected]

lifetechnologies.com

dna isolation kit - thermo fisher...

Documents