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DIG Application Manual for Filter Hybridization.Indb 778/779 DIG Hyb ManualCover_1AK 30.07.2008 14:32 Uhr Seite 3 C M Y CM MY CY CMY K Probedruck DIG_Filter_ManualCover_RZ 01.08.2008 15:00 Uhr Seite 4 C M Y CM MY CY CMY K Intended use Our preparations are exclusively intended to be used in life science research applications.* They must not be used in or on human beings since they were neither tested nor intended for such utilization. Preparations with hazardous substances Our preparations may represent hazardous substances to work with. The dangers which, to our knowledge, are involved in the handling of these preparations (e.g., harmful, irritant, toxic, etc.), are separately mentioned on the labels of the packages or on the pack inserts; if for certain preparations such danger references are missing, this should not lead to the conclusion that the corresponding preparation is harmless. All preparations should only be handled by trained personnel. Preparations of human origin The material has been prepared exclusively from blood that was tested for Hbs antigen and for the presence of antibodies to the HIV-1, HIV-2, HCV and found to be negative. Nevertheless, since no testing method can offer complete assurance regarding the absence of infectious agents, products of human origin should be handled in a manner as recommended for any potentially infectious human serum or blood specimen. Liability The user is responsible for the correct handling of the products and must follow the instructions of the pack insert and warnings on the label. Roche Diagnostics shall not assume any liability for damages resulting from wrong handling of such products. Because of its outstanding tactile sensitivity, the Red-Eyed-Tree Frog (Agalychnis callidryas) was chosen to represent the high sensitivity of the DIG System. * exeption: instruments specifically intended for in-vitro diagnostic use. Impressum © 2008 by Roche Diagnostics GmbH Editorial Management: Doris Eisel Oliver Seth, PH.D. Stefanie Grünewald-Janho Bettina Kruchen, PH.D. Barbara Rüger, PH.D. Art Direction and Design: Designgruppe Fanz & Neumayer Schifferstadt Layout and Typesetting: ACTIVE ARTWARE Gruppe Saarbrücken Probedruck DIG Application Manual for Filter Hybridization __DIGDIG AApplicationpplication MManualanual fforor FFilterilter HHybridization.indbybridization.indb 1 005.08.20085.08.2008 110:20:490:20:49 __DIGDIG AApplicationpplication MManualanual fforor FFilterilter HHybridization.indbybridization.indb 2 005.08.20085.08.2008 110:20:560:20:56 Table of Contents Chapter 1 Introduction Introduction to the DIG System and the DIG Application Manual .......................8 An Overview of the DIG Nonradioactive Nucleic Acid Labeling and Detection System ............................................................................................................8 An Overview of the DIG Application Manual ............................................................12 Chapter 2 DIG Basics 1. Introduction ............................................................................................................. 16 2. DIG Labeling ........................................................................................................... 17 2.1 Overview of Nonradioactive DIG Labeling Methods ..................................18 2.2 Comparison of PCR Labeling Random Primed Labeling and RNA Labeling ...................................................................................................20 3. Evaluation of Probe Labeling Effi ciency ................................................... 23 3.1 Overview of Direct Detection Procedure .........................................................23 3.2 Analysis of PCR-labeled Probes by Gel Electrophoresis ........................... 25 4. Electrophoresis of Target Nucleic Acids on Agarose Gels ............ 26 4.1 Amount of Target Nucleic Acid To Load ..........................................................26 4.2 Pouring and Running the Agarose Gel ............................................................26 5. Blot Transfer of Target Nucleic Acids to a Membrane ..................... 28 5.1 Comments on Blot Membranes..........................................................................30 6. Hybridization of Probe to Target ...................................................................31 6.1 Concentration of DIG-labeled Probe During Hybridization .....................31 6.2 Determining an Optimal Hybridization Temperature ..................................31 6.3 Components of the Hybridization Buffer .........................................................33 6.4 Hybridization Time Required ................................................................................34 7. Stringent Washes of Blot .................................................................................. 35 8. Detection of Probe-Target Hybrids..............................................................36 9. Stripping and Reprobing the Membrane ................................................38 10. Equipping a DIG Labeling and Detection Lab ......................................40 11. Product Literature and Support Material ................................................ 43 12. DIG Product Selection Guide and Ordering Information ...............44 3 __DIGDIG AApplicationpplication MManualanual fforor FFilterilter HHybridization.indbybridization.indb 3 005.08.20085.08.2008 110:20:560:20:56 Table of Content Chapter 3 Procedures for Nonradioactive 1. Introduction .............................................................................................................54 Labeling and Detection 2. Techniques for DIG Labeling of Hybridization Probes ..................... 55 2.1 Random Primed Labeling of DNA Probes (High Yield Method) ............ 57 2.1.1 Materials Required for Random Primed Labeling .......................................58 2.1.2 Procedures ..................................................................................................................58 2.1.3 Getting the Best Results from Random Primed Labeling .........................62 2.2 PCR Labeling of DNA Probes..............................................................................64 2.2.1 Materials required for PCR Labeling ................................................................64 2.2.2 Procedures .................................................................................................................. 65 2.2.3 Getting the Best Results from PCR Labeling ................................................ 70 2.2.4 Typical Results with DIG-labeled Probes Generated by PCR.................. 73 2.3 Transcriptional Labeling of RNA Probes .........................................................74 2.3.1 Materials Required for RNA Probe Labeling .................................................75 2.3.2 Procedures .................................................................................................................. 77 2.3.3 Getting the Best Results from Transcriptional Labeling ............................81 2.3.4 Typical Results with DIG-labeled RNA probes .............................................83 2.4 DIG Oligonucleotide Labeling .............................................................................84 2.5 Estimation of Probe Yield by the Direct Detection Procedure ................85 2.5.1 Materials Required for Direct Detection Procedure ...................................85 2.5.2 Procedures ..................................................................................................................86 3. Techniques for Hybridization of DIG-labeled Probes to a Blot ...94 3.1 Hybridization of DNA Probes to a Southern Blot .........................................94 3.1.1 Materials Required for Electrophoresis, Blotting, and Hybridization ....95 3.1.2 Procedures ..................................................................................................................96 3.1.3 How To Use DIG-labeled Oligonucleotide Probes To Detect DNA Targets on a Southern Blot .................................................102 3.2 Hybridization of RNA Probes to a Northern Blot .......................................103 3.2.1 Materials Required for Electrophoresis, Blotting, and Hybridization ..103 3.2.2 Procedures ................................................................................................................105 3.2.3 How To Use DIG-labeled DNA Probes To Detect RNA Targets on a Northern Blot ...........................................................................................................111 3.3 Getting the Best Results from Blots ................................................................112 3.3.1 Critical Hints about Electrophoresis, Blotting, and Hybridization ........112 3.3.2 Troubleshooting the Hybridization Blot ........................................................114 4 DIG Application Manual for Filter Hybridization __DIGDIG AApplicationpplication MManualanual fforor FFilterilter HHybridization.indbybridization.indb 4 005.08.20085.08.2008 110:20:560:20:56 Table of Content Chapter 3 Procedures for Nonradioactive 4. Techniques for Detection of Hybridization Probes on a Blot .....115 Labeling and Detection 4.1 Chemiluminescent Methods for Detection of Probes on a Blot ...........115 4.1.1 Materials Required for Chemiluminescent Detection ..............................116 4.1.2 Procedure ..................................................................................................................116 4.1.3 Troubleshooting the Chemiluminescent Detection
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