Gel Electrophoresis: Nucleic Acids / Edition 1

by Dr Robin Martin

Overview

Through its clear presentation of the basic concepts, Gel Electrophoresis: Nucleic Acids breaks new ground by describing the principles of the technique without resorting to complicated protocols and recipes.

Product Details

ISBN-13:	9781872748283
Publisher:	Taylor & Francis
Publication date:	06/15/1996
Series:	Introduction to Biotechniques Series
Pages:	192
Product dimensions:	6.12(w) x 9.19(h) x (d)

About the Author

Group Leader Wound Healing Research, Blond Mcindoe Centre, Queen Victoria Hospital, East Grinstead, West Sussex RH19 3DZ, UK.

Table of Contents

Abbreviations — Preface — PART 1: BASIC PRINCIPLES AND METHODS — 1. Introduction: the Variety and Forms of Nucleic Acids — Overview — Electrophoresis and the properties of nucleic acids — The variety and forms of nucleic acid — References — 2. The Theory of Nucleic Acid Electrophoresis — The movement of nucleic acids in liquids and in gels — Electric currents and buffer solutions — Nucleic acids in solution — Nucleic acids in gels — The movement of nucleic acids through gels in constant electric fields — Ogston sieving — Reptation — Migration as rigid rods — The movement of nucleic acids through gels in pulsed electric fields — References — 3. The Electrophoresis of Native and Denatured Nucleic Acids — The control of base pairing — Physical and chemical denaturants for nucleic acid gel electrophoresis — Temperature — Alkaline conditions — Methyl mercuric hydroxide, glyoxal and formaldehyde: denaturants for RNA in agarose gels — Urea and formamide — The binding of proteins to nucleic acids during gel electrophoresis — References — 4. The Choice of Format: Horizontal or Vertical, Agarose or Polyacrylamide? — Apparatus for horizontal and vertical gels — Apparatus for horizontal agarose gels — Apparatus for vertical polyacrylamide gels — Power supplies for nucleic acid gel electrophoresis — Agarose gel media for nucleic acid electrophoresis — Modified agarose preparations — Low melting point agarose — Higher strength agarose — High-sieving agarose — Visigel"' — Ready-made agarose gels — Polyacrylamide gel media for nucleic acid gel electrophoresis — Modified polyacrylamide formulations — Ready-made polyacrylamide gels — Wedge shaped gels — Toxicity of the components of polyacrylamide gels — References — 5. The Detection of Nucleic Acids Following Electrophoretic Separation — Overview — The principles of nucleic acid detection — Binding with fluorescent dyes — Labeling with radioactive nucleotides — Labeling with fluorescent nucleotides — Labeling nucleic acids with specific proteins — Indirect DNA-protein coupling — Direct DNA-protein coupling — Conventional staining — Nucleic acid detection in situ and after transfer to membranes — Summary of nucleic acid detection methodology — References — PART 2: TECHNIQUES AND APPLCATIONS — 6. Guide to Techniques and Applications — Matching the molecule to the technique — Strategic considerations: avoiding elementary mistakes — 7. Nondenaturing Agarose Gel Electrophoresis — Buffers for nondenaturing agarose gels — The resolving power of agarose: altering the gel concentration; altering the field strength — Estimating the length of unknown fragments using semi-log plots — Solving a separation problem: resolving the 5148 and 4973 hp bands in the HindIII-EcoRI restriction digest of phage A DNA — Alternative methods for staining with ethidium bromide — Passive diffusion; can agarose gels be left safely overnight before photography? — Amounts of DNA: estimating unkown quantities and before photography? — Estimating unknown quantities — Overloading and underloading a gel — DNA conformation and the mobility of molecules in agarose gels — Heating A DNA markers — A note on taking pictures — Recording the position of marker bands when DNA is transferred to a membrane — Research applications: overview — Research application: Nondenaturing agarose gel electrophoresis: Southern blotting — Background — Procedure — Interpretation — References — 8. Denaturing Agarose Gel Electrophoresis — Research application. Denaturing gel electrophoresis of single-stranded RNA molecules: Northern blotting — Background — Procedure — Interpretation — References — 9. Pulsed Field Agarose Gel Electrophoresis — The principles of pulsed field technology — Pulsed field electrode geometry — Sample preparations for pulsed field gels — Research application. Pulsed field agarose gels for genome mapping — Background — Procedure — Interpretation — References — 10. Nondenaturing Polyacrylamide Gel Electrophoresis — Overview — Research application. High-resolution separation of double-stranded DNA fragments — Background — Procedure — Interpretation — Single- and double-strand conformational polymorphisms: SSCP and DSCP — The principle of using gel electrophoretic conformational polymorphisms to detect mutations — Research application. Nondenaturing polyacrylamide gel electrophoresis: using SSCP analysis to detect mutations — Bandshift or gel retardation assays — The principles of the bandshift assay — Research application. Bandshifting assays — References — 11. Denaturing Polyacrylamide Gel Electrophoresis — Overview — DNA sequencing — The principles of DNA sequencing — Research application. The use of a dGTP base analog to overcome a sequencing gel compression artifact — DNA footprinting — The principle of DNA footprinting — Research application. DNA footprinting — RNase protection assays — The principle of the RNase protection assay — Research application. RN ase protection assay — Nuclease S1 protection assays — The principle of nuclease S1 protection assays — Research application. Nuclease S1 protection assay — Primer extension assays — The principle of the primer extension assay — Research application. Primer extension assays — References — Appendices — Appendix A: Glossary — Appendix B: Suppliers — Index.