En QIAGEN Genomic DNA Handbook

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Text of En QIAGEN Genomic DNA Handbook

  • August 2001

    QIAGEN Genomic DNA Handbook

    ForBloodCultured cellsTissueMouse tailsYeastBacteria (Gram-negative and some Gram-positive)

  • QIAGEN DistributorsPlease see the last page for contact information for your local QIAGEN distributor.

    Australia QIAGEN Pty Ltd PO Box 25 Clifton Hill Victoria 3068ABN 75 072 382 944 Orders 03-9489-3666 Fax 03-9489-3888 Technical 1-800-243-066

    Canada QIAGEN Inc. 2900 Argentia Road Unit 23 Mississauga Ontario L5N 7X9Orders 800-572-9613 Fax 800-713-5951 Technical 800-DNA-PREP (800-362-7737)

    France QIAGEN S.A. 3 avenue du Canada LP 809 91974 Courtaboeuf CedexOrders 01-60-920-920 Fax 01-60-920-925 Technical 01-60-920-930

    Germany QIAGEN GmbH Max-Volmer-Strae 4 40724 HildenOrders 02103-29-12000 Fax 02103-29-22000 Technical 02103-29-12400

    Italy QIAGEN S.p.A. Via Grosio, 10/10 20151 MilanoOrders 02-33430411 Fax 02-33430426 Technical 02-33430414

    Japan QIAGEN K.K. Forefront Tower II 13-1, Kachidoki 3 Chome Chuo-ku, Tokyo 104-0054www.qiagen.co.jp Telephone 03-5547-0811 Fax 03-5547-0818 Technical 03-5547-0811

    Switzerland QIAGEN AG Auf dem Wolf 39 4052 BaselOrders 061-319-30-30 Fax 061-319-30-33 Technical 061-319-30-31

    UK QIAGEN Ltd. Boundary Court Gatwick Road Crawley West Sussex, RH10 9AXOrders 01293-422-911 Fax 01293-422-922 Technical 01293-422-999

    USA QIAGEN Inc. 28159 Avenue Stanford Valencia CA 91355Orders 800-426-8157 Fax 800-718-2056 Technical 800-DNA-PREP (800-362-7737)

    www.qiagen.com

    QIAGEN Companies

    QIAGEN Worldwide

    19992001 QIAGEN, all rights reserved.

  • ContentsKit Contents 5

    Storage Conditions 6

    Product Use Limitations 6

    Product Warranty and Satisfaction Guarantee 7

    Technical Assistance 7

    Safety Information 8

    Introduction 9

    The QIAGEN Principle 11

    The QIAGEN Genomic DNA Purification Procedure 11

    Sample volumes and capacities of QIAGEN Genomic-tips 11Preparation of the lysate 12Enzymes 13QIAGEN Protease and Proteinase K 13Setup of QIAGEN Genomic-tips 15Capacity of QIAGEN Genomic-tips 15DNA binding and washing on QIAGEN Genomic-tips 15Desalting and concentration 16Analytical gel analysis 16

    Part I: Sample Preparation and Lysis Protocols

    Blood 17

    Storage of blood samples 17Recommended cell densities 17

    A. Isolation of genomic DNA from whole blood of healthy donors 18B. Isolation of genomic DNA from whole blood of unhealthy donors 18

    1. Leukocyte concentrations higher than 1 x 107/ml 182. Leukocyte concentrations lower than 5 x 106/ml 18

    C. Isolation of genomic DNA from buffy-coat preparations 18D. Isolation of genomic DNA from lymphocyte preparations 19

    1. Ficoll gradient preparation of lymphocytes 192. Dextran sedimentation 19

    Sample Preparation and Lysis Protocol for Blood 20

    Cell Cultures 23

    Storage of cell samples 23Recommended cell densities 23

    A. Cells grown in suspension 23B. Cells grown in a monolayer 24

    QIAGEN Genomic DNA Handbook 08/2001 3

  • Sample Preparation and Lysis Protocol for Cell Cultures 25

    Tissue 28

    Storage of tissue samples 28Recommended sample amounts 28

    Sample Preparation and Lysis Protocol for Tissue 29

    Yeast 32

    Recommended culture volumes 32

    Sample Preparation and Lysis Protocol for Yeast 34

    Bacteria 38

    Recommended culture volumes 38

    Sample Preparation and Lysis Protocol for Bacteria 40

    Part II: Genomic-tip Protocol

    Protocol for Isolation of Genomic DNA from Blood, Cultured Cells, Tissue, Yeast, or Bacteria 44

    Determination of yield, purity, and length of the DNA 46Analytical gel 47

    Special Applications 48

    Purification of genomic DNA prepared by other methods 48Preparation of genomic DNA for PCR applications 48

    Troubleshooting Guide 50

    Appendix A 55

    Preparation of buffers 56Preparation of culture media 57Preparation of enzyme stock solutions 57

    Appendix B 59General information about QIAGEN Anion-Exchange Resin 59Purity and biological activity 60Capacity and recovery 60Stability 60Buffers 61

    Ordering Information 62

    QIAGEN Distributors 67

    Experienced User Protocols Available on request

    QIAGEN Genomic DNA Handbook 08/20014

  • QIAGEN Genomic DNA Handbook 08/2001 5

    Kit Contents

    QIAGEN Blood & Cell Culture DNA Kit Mini Midi Maxi

    Catalog No. 13323 13343 13362

    QIAGEN Genomic-tip 20/G 25

    QIAGEN Genomic-tip 100/G 25

    QIAGEN Genomic-tip 500/G 10

    Tip Holders 6 5 5

    Buffer C1 40 ml 170 ml 250 ml

    Buffer G2* 60 ml 260 ml 260 ml

    Buffer QBT 60 ml 110 ml 110 ml

    Buffer QC 120 ml 410 ml 330 ml

    Buffer QF 60 ml 140 ml 170 ml

    QIAGEN Protease 1 vial 2 vials 2 vials

    Handbook 1 1 1

    Genomic DNA Buffer Set (for 75 mini, 25 midi, or10 maxi preparations)

    Catalog No. 19060

    Buffer C1 250 ml

    Buffer Y1 160 ml

    Buffer B1 120 ml

    Buffer B2* 50 ml

    Buffer G2* 260 ml

    Buffer QBT 110 ml

    Buffer QC 410 ml

    Buffer QF 170 ml

    Handbook 1

    * Not compatible with disinfection reagents containing bleach. Contains guanidine HCl, which is an irritant.Take appropriate safety measures and wear gloves when handling.

    Resuspension volume 1.4 ml

    Note: QIAGEN Protease, Proteinase K, or other required enzymes must be purchased separately as specified.

    QIAGENGenomic-tip 20/G 100/G 500/G

    Catalog No. 10223 10243 10262

    Genomic-tip 25 25 10

    Tip Holders 6 5 5

    Handbook 1 1 1

  • Storage ConditionsQIAGEN Genomic-tips should be stored dry and at room temperature. They can bestored for at least two years without showing any reduction in performance, capacity, orquality of separation.

    Buffer C1 and Buffer Y1 should always be stored at 28C. All other buffers may be storedat either 28C or at room temperature (1525C). Long-term storage at 28C is recommended.

    Lyophilized QIAGEN Protease can be stored at room temperature (1525C) for up to6 months without any decrease in performance. For storage longer than 6 months or ifambient temperatures constantly exceed 25C, QIAGEN Protease should be stored dryat 28C.

    Reconstituted QIAGEN Protease is stable for 2 months when stored at 28C. Incubatingthe QIAGEN Protease stock solution at room temperature for prolonged periods of timeshould be avoided. Storage at 20C will prolong its life, but repeated freezing andthawing should be avoided. Dividing the solution into aliquots and storage at 20C isrecommended.

    QIAGEN Proteinase K is stable at room temperature for at least one year. For longer-termstorage, we recommend storing at 28C.

    Dissolved lysozyme, lysostaphin, lyticase, and zymolase must be stored at 20C, butrepeated freezing and thawing should be avoided. When storage at 20C is necessary,dividing the enzyme solutions into aliquots is recommended.

    Product Use LimitationsSome QIAGEN products may be used in clinical diagnostic laboratory systems after thelaboratory has validated their complete systems as required by CLIA 88 regulations inthe U.S. or equivalents in other countries. All due care and attention should be exercisedin the handling of the products. We recommend all users of QIAGEN products to adhereto the NIH guidelines that have been developed for recombinant DNA experiments, orto other applicable guidelines.

    QIAGEN Genomic DNA Handbook 08/20016

  • Product Warranty and Satisfaction GuaranteeQIAGEN guarantees the performance of all products in the manner described in ourproduct literature. The purchaser must determine the suitability of the product for itsparticular use. Should any product fail to perform satisfactorily due to any reason otherthan misuse, QIAGEN will replace it free of charge or refund the purchase price. Wereserve the right to change, alter, or modify any product to enhance its performance anddesign. If a QIAGEN product does not meet your expectations, simply call your localTechnical Service Department. We will credit your account or exchange the product as you wish.

    A copy of QIAGEN terms and conditions can be obtained on request, and is alsoprovided on the back of our invoices. If you have questions about product specificationsor performance, please call QIAGEN Technical Services or your local distributor (seeinside front cover).

    Technical AssistanceAt QIAGEN we pride ourselves on the quality and availability of our technical support.Our Technical Service Departments are staffed by experienced scientists with extensivepractical and theoretical expertise in molecular biology and the use of QIAGENproducts. If you have any questions or experience any difficulties regarding QIAGENGenomic-tips or QIAGEN products in general, please do not hesitate to contact us.

    QIAGEN customers are a major source of information regarding advanced or specializeduses of our products. This information is helpful to other scientists as well as to theresearchers at QIAGEN. We therefore encourage you to contact us if you have anysuggestions about product performance or new applications and techniques.

    For technical assistance and more information please call one of the QIAGEN TechnicalService Departments or local distributors (see inside front cover).

    QIAGEN Genomic DNA Handbook 08/2001 7

  • Safety InformationWhen working with chemicals, always wear a suitable lab coat, disposable gloves, andprotective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDFformat at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDSfor each QIAGEN kit and kit components.

    CAUTION: DO NOT add bleach or acidic solutions directly to

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