Hybridization Membranes and Supports 413 What Are the Criteria for Selecting a Support for Your Hybridization Experiment? 413 Which Membrane Is Most Appropriate for Quantitative Experiments? 4l7 What Are the indicators of a functional membrane? 4l7 Can Nylon and Nitrocellulose Membranes Be Sterilized? Nucleic acid transfer 4|8 What Issues affect the transfer of nucleic acid from Agarose Gels 418 Should Membranes Be Wet or Dry Prior to Use What Can You Do to Optimize the Performance of Colony and plaque Transfers? Crosslinking Nucleic Acids 422 What Are the Strengths and Limitations of Common Crosslinking Strategies? 422 What Are the Main Problems of Crosslinking? 423 What's the Shelf Life of a membrane whose Target DNA The Hybridization Reaction 424 How Do You Determine an Optimal Hybridization Te 424 What Range of Probe Concentration Is Acceptable?... 425 What Are Appropriate Pre-hybridization Times? 426 How Do You Determine Suitable Hybridization Times?.. 426 What Are the Functions of the Components of a Typical Hybridization Buffer? 427 What to Do before You Develop a New Hybridization Buffer formulation? 430 What Is the Shelf Life of Hybridization Buffers and opPonents 43l What Is the Best Strategy for Hybridization of Multiple Membranes 432 Is Stripping Always Required Prior to Reprobing? 432 What Are the Main Points to Consider When Reprobing Blots? 433 How Do You optimize Wash Steps? How Do You Select the Proper Hybridization 435 Detection by Autoradiography Film 436 How Does an Autoradiography Film Function? 436 What Are the Criteria for Selecting Autoradiogaphy Film? 438 Why Expose Film to a blot 440 400 Herzer and EnglertHybridization Membranes and Supports . . . . . . . . . . . . . . . . . . 413 What Are the Criteria for Selecting a Support for Your Hybridization Experiment? . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Which Membrane Is Most Appropriate for Quantitative Experiments? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 What Are the Indicators of a Functional Membrane? . . . . . 417 Can Nylon and Nitrocellulose Membranes Be Sterilized? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Nucleic Acid Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 What Issues Affect the Transfer of Nucleic Acid from Agarose Gels? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 Should Membranes Be Wet or Dry Prior to Use? . . . . . . . . 420 What Can You Do to Optimize the Performance of Colony and Plaque Transfers? . . . . . . . . . . . . . . . . . . . . . . . 421 Crosslinking Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 What Are the Strengths and Limitations of Common Crosslinking Strategies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 What Are the Main Problems of Crosslinking? . . . . . . . . . . . 423 What’s the Shelf Life of a Membrane Whose Target DNA Has Been Crosslinked? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 The Hybridization Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 How Do You Determine an Optimal Hybridization Temperature? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 What Range of Probe Concentration Is Acceptable? . . . . . . 425 What Are Appropriate Pre-hybridization Times? . . . . . . . . . 426 How Do You Determine Suitable Hybridization Times? . . . 426 What Are the Functions of the Components of a Typical Hybridization Buffer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 What to Do before You Develop a New Hybridization Buffer Formulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 What Is the Shelf Life of Hybridization Buffers and Components? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 What Is the Best Strategy for Hybridization of Multiple Membranes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Is Stripping Always Required Prior to Reprobing? . . . . . . . . 432 What Are the Main Points to Consider When Reprobing Blots? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 How Do You Optimize Wash Steps? . . . . . . . . . . . . . . . . . . . 434 How Do You Select the Proper Hybridization Equipment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Detection by Autoradiography Film . . . . . . . . . . . . . . . . . . . . . . 436 How Does an Autoradiography Film Function? . . . . . . . . . . . 436 What Are the Criteria for Selecting Autoradiogaphy Film? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 Why Expose Film to a Blot at -70°C? . . . . . . . . . . . . . . . . . 440 400 Herzer and Englert