核苷酸杂交技术大全
Planning a Hybridization Experiment ... ... ... ... ... .. ... . 401
The Importance of Patience .. ... ... ... ... ... ... .. ... . 401
What Are Your Most Essential Needs? .. ... ... ... .. ... . 401
Visualize Your Particular Hybridization Event ... ... .. ... . 401
Is a More Sensitive Detection System Always Better? . ... . 403
What Can You Conclude from Commercial
Sensitivity Data? . ... ... ... ... ... ... ... ... ... .. ... . 403
Labeling Issues ........................................ 403
Which Labeling Strategy Is Most Appropriate for
Your Situation? ... ... ... ... ... ... ... ... ... ... .. ... . 403
What Criteria Could You Consider When Selecting
a Label? . .. ... ... ... ... ... ... ... ... ... ... ... .. ... . 405
Radioactive and Nonradioactive Labeling Strategies
Compared ... .. ... ... ... ... ... ... ... ... ... ... ... .. ... . 409
What Are the Criteria for Considering Direct over
Indirect Nonradioactive Labeling Strategies? .. ... .. ... . 410
What Is the Storage Stability of Labeled Probes? .. ... .. .. 411
Should the Probe Previously Used within the
Hybridization Solution of an Earlier Experiment Be
Applied in a New Experiment? . ... ... ... ... ... .. ... . 412
How Should a Probe Be Denatured for Reuse? . ... .. ... . 412
Is It Essential to Determine the Incorporation Efficiency
of Every Labeling Reaction? . ... ... ... ... ... ... .. ... . 412
Is It Necessary to Purify Every Probe? .. ... ... ... .. ... . 413
399
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? . ... ... ... .. ... ... ... ... ... ... ... ... . 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
Helpful Hints When Working With Autoradiography
Film . ... ... .. ... ... ... ... ... ... ... ... ... ... ... .. . 441
Detection by Storage Phosphor Imagers ... ... ... ... ... .. . 441
How Do Phosphor Imagers Work? .. ... ... ... ... ... ... . 441
Is a Storage Phosphor Imager Appropriate for Your
Research Situation? .. ... ... ... ... ... ... ... ... ... .. . 441
What Affects Quantitation? ... ... ... ... ... ... ... ... .. . 443
What Should You Consider When Using Screens? .. ... .. . 445
How Can Problems Be Prevented? ... ... ... ... ... ... .. . 447
Troubleshooting . .. ... ... ... ... ... ... ... ... ... ... ... .. . 448
What Can Cause the Failure of a Hybridization
Experiment? .. ... ... ... ... ... ... ... ... ... ... ... .. . 448
Bibliography . ... .. ... ... ... ... ... ... ... ... ... ... ... .. . 453
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