Nucleic Acid Blotting

CHAPTER I How it all got started -- 1. The development of blotting and hybridization techniques -- 1.1 Southern blotting -- 1.2 Northern blotting -- 1.3 Further advances -- 2. This book -- 3. Further reading -- 4. Laboratory safety -- 5. References -- CHAPTER 2 Southern blotting I: electrophoresis of DNA -- 1. Different sources of DNA -- 1.1 Genomic DNA -- 1.2 Plasmid DNA -- 1.3 Bacteriophage and cosmid DNA -- 1.4 YACDNA -- 1.5 PCR product DNA -- 2. Before you load the DNA on to the gel -- 3. Preparing and running the gel -- 3.1 Agarose -- 3.2 Electrophoresis buffers -- 3.3 Casting the gel -- 3.4 Assembling the gel electrophoresis tank -- 3.5 Loading your samples -- 3.6 Running the gel -- 3.7 Visualizing the DNA with UV radiation -- 3.8 Photographing your gel -- 3.9 Interpretation of gels -- 3.9.1 Genomic DNA -- 3.9.2 Cloned DNA -- 3.10 What can go wrong with electrophoresis? -- 4. Further reading -- 5. References -- CHAPTER 3 Southern blotting II: performing the blot -- I. Uni-directional capillary blotting on to a single membrane at neutral pH -- 1.1 Preparing the gel for blotting -- 1.1.1 Partial depurination -- 1.1.2 Denaturation -- 1.1.3 Neutralization -- 1.2 Assembling the blot -- 1.2.1 Cutting and preparing the membrane -- 1.2.2 Assembly and blotting -- 1.3 Dismantling the blot -- 1.4 Checking the efficiency of transfer -- 1.5 Fixing the DNA to the membrane -- 1.5.1 UV treatment -- 1.5.2 Drying -- 1.6 Storing membranes before hybridization -- 2. Capillary blotting on to multiple membranes at neutral pH -- 2.1 Uni-directional capillary blotting on to several membranes -- 2.2 Bi-directional capillary blotting -- 3. Capillary blotting at alkaline pH -- 4. Other methods of blotting -- 4.1 Electrophoretic transfer ( electro blotting) -- 4.2 Vacuum blotting and positive-pressure blotting -- 4.3 Which method of blotting should you use? -- 5. Further reading -- 6. References -- CHAPTER 4 Electrophoresis of RNA and northern blotting -- 1. How does northern blotting differ from Southern blotting? -- 2. What information can a northern blot give? -- 3. Comparing the levels of an mRNA species in different cell types -- 3.1 Equal loading -- 3.2. Quantitation -- 3.2.1 Scintillation counting of excised bands -- 3.2.2 Scanning densitometry -- 3.2.3 Phosphorimagery -- 4. Gel electrophoresis of RNA samples -- 4.1 Gel systems -- 4.2 Formaldehyde gels -- 4.2.1 Preparing formaldehyde gels -- 4.2.2 Preparing RNA samples for formaldehyde gels -- 4.2.3 How much RNA should be loaded? -- 4.2.4 Running formaldehyde gels -- 4.3 Glyoxal gels -- 4.3.1 Preparing glyoxal gels -- 4.3.2 Preparing RNA samples for glyoxal gels -- 4.3.3 Running glyoxal gels -- 4.4 Molecular size markers -- 4.4.1 28S and 18S rRNA size markers -- 4.4.2 Other RNA size markers -- 5. Blotting the gel -- 6. Further reading -- 7. References -- CHAPTERS Dot and slot blotting -- 1. What are dot blots and slot blots -- 1.1 Why do a DNA dot /slot blot? -- 1.2 Why do an RNA dot /slot blot? -- 2. How to perform a dot /slot blot -- 2.1 Preparing the sample -- 2.1.1 How much DNA or RNA should you load? -- 2.1.2 DNA samples -- 2.1.3 RNA samples -- 2.2 Preparing the membrane -- 2.3 Assembling the manifold -- 2.4 Applying the sample -- 2.5 Blotting -- 2.6 Processing the membrane -- 3. Quantitation of dot /slot blots and interpretation of results -- 4. Limitations of dot /slot blotting -- 5. Further reading -- 6. References -- CHAPTER 6 Plaque and colony screening -- 1. Screening bacteriophage 11, plaques by the Benton and Davis method -- 1.1 Plating -- 1.1.1 Petri dishes -- 1.1.2 Base agar -- 1.1.3 Top agarose -- 1.1.4 Plating cells -- 1.1.5 Bacteriophage -- 1.1.6 Plating -- 1.2 Taking membrane lifts -- 1.2.1 Getting the membranes ready -- 1.2.2 Taking the first membrane lift -- 1.2.3 Taking the second membrane lift -- 1.2.4 Storing the plates -- 1.3 Treating membranes before hybridization -- 1.3.1 Denaturation -- 1.3.2 Neutralization -- 1.3.3 Treatment with 2 x SSC -- 1.3.4 Fixing the DNA to the membrane -- 1.4 A brief note about hybridization probes -- 1.5 Orientating the membranes and X-ray film before autoradiography -- 1.6 Identifying hybridization signals after autoradiography -- 1. 7 Picking plaques -- 1.8 Further rounds of screening -- 2. Determining the titre of a bacteriophage "A suspension -- 3. Screening bacterial colonies by the Grunstein-Hogness method -- 3.1 Plating -- 3.2 Making replica membranes -- 3.2.1 Making the first replica -- 3.2.2 Making the second replica -- 3.2.3 What to do with the master membrane while you are screening the replicas -- 3.3 Treating membranes before hybridization -- 3.4 Another brief note about hybridization probes -- 3.5 Orientating the membranes and X-ray film before autoradiography -- 3.6 Picking colonies -- 4. Further reading -- 5. References -- CHAPTER 7 Filters and membranes -- 1. The advantages and disadvantages of nitrocellulose filters and nylon membranes? -- 1.1 Nylon membranes are physically strong -- 1.2 DNA and RNA bind covalently to nylon membranes -- 1.3 Nylon membranes have a high nucleic acid binding capacity -- 1.4 Nylon membranes are hydrophilic -- 1.5 Nylon membranes retain"their size and shape at high temperatures -- 1.6 Nylon membranes are not inflammable -- 1. 7 Nylon membranes do not require solutions of high ionic strength to bind nucleic acids efficiently -- 1.8 Nylon membranes may give higher backgrounds than nitrocellulose filters, but this can be overcome easily -- 1.9 Nylon membranes are sometimes 'single-sided' -- 1.10 Nylon membranes and nitrocellulose filters must be handled with care -- 2. Which membrane should be used? -- 3. References -- Glossary -- Index.

Biography

The Medical Molecular Biology Unit Department of Molecular Pathology University College London Medical School The Windeyer Building, Cleveland Street, London WI P 6DB.