Robin Martin

Gel Electrophoresis: Nucleic Acids (eBook, ePUB)

• Format: ePub

Produktbeschreibung

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.

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Produktdetails

• Verlag: Taylor & Francis
• Seitenzahl: 192
• Erscheinungstermin: 26. August 2020
• Englisch
• ISBN-13: 9781000140927
• Artikelnr.: 60062259

Autorenporträt

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

Inhaltsangabe

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.