See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/316976812

Biological Tools & Techniques (A textbook for UG/PG students

of Life Sciences)

Book · January 2017

CITATIONS READS
0 73,892

1 author:

Ananta Swargiary
Bodoland University
65 PUBLICATIONS 663 CITATIONS

SEE PROFILE

All content following this page was uploaded by Ananta Swargiary on 16 May 2017.

The user has requested enhancement of the downloaded file.

Publisher: Kalyani Publishers, New Delhi
Price: 195/-
 CONTENTS

Dedication
About the author
Preface and acknowledgment
List of reviewers
Contents

CHAPTER-1: HISTOLOGY AND MICROTOMY

Introduction
Types of Histological Preparations
Microtomy
Types of Microtomes
Microtome Knives
Paraffin Method of Sectioning
Problems and Solutions of Paraffin Microtomy
Frozen Method of Sectioning
The Cryostat (Ultramicrotome)
Freezing of fresh unfixed tissue
Fixed tissue and the cryostat
Cryostat Sectioning
Ultracryotomy
Freeze drying and freeze substitution
Cryopreservation
Slow Freeze Drying
Vitrification
Cryoprotectant
Germplasm cryopreservation
Cryopreservation of Mammalian Semen (Sperm)
Cryopreservation of Mammalian Oocytes (Eggs)
Factors affecting on oocyte cryopreservation

CHAPTER-2: TISSUE FIXATION AND PROCESSING

Introduction
Properties of a good fixative
Types of Fixatives
Physical Methods of Fixation
Chemical Methods of Fixation
Non-coagulant cross-linking fixatives
Cross linking fixatives for electron microscopy
Factors affecting the quality of fixation
Tissue processing
Principles of tissue processing
Basic steps of tissue processing
Commonly used staining solutions and its formulae

CHAPTER-3: MICROSCOPY
Introduction
Basic principle of Microscopy
Resolving power of microscope
Types of microscope
Simple Microscope
Component parts of a simple microscope
Compound Microscope
Light Microscopy
Bright field microscope
Dark field microscope
Phase contrast microscope
Working principle of phase contrast microscope
Structural Component of Phase Contrast Microscope
Fluorescence Microscope
Principle of fluorescence
Components of a fluorescence microscope
Confocal microscopy
Principle of Confocal Microscope
Types of confocal microscopes
Sample Preparation of Light microscopy
Electron Microscopy
Types of electron microscope
Transmission Electron Microscopy
Working principle of TEM
Sample preparation for TEM
Scanning Electron Microscopy
Working principle of SEM
Resolution of SEM
Magnification in SEM
Sample preparation for SEM
Advantages and Disadvantages of SEM
CHAPTER-4: CENTRIFUGATION
Introduction
Principle of Centrifugation
Centrifugal force
Principles of Sedimentation
Svedberg co-efficient
Basic component of a centrifuge machine
Types of centrifuges and their uses
Differential Centrifugation
Density Gradient Centrifugation
Sub-cellular Fractionation

CHAPTER-5: ELECTROPHORETIC TECHNIQUES

Introduction
Theory
Basic Principle of electrophoresis
Effect of buffer on electrophoretic movement
Instrumentation Setup of Electrophoresis
Supporting Medium
Type of Electrophoresis
Boundary electrophoresis
Paper electrophoresis
Gel electrophoresis
Polyacrylamide gel electrophoresis
Disc electrophoresis
SDS-PAGE
Analysis of the Gels
Native Page
Gradient Gels or Pore Gradient Electrophoresis
Isoelectric Focusing
2-Dimensional Polyacrylamide Gel Electrophoresis
Electrophoresis of Nucleic Acids
Agarose Gel Electrophoresis
Pulsed-Field Gel Electrophoresis
Western Blotting

CHAPTER-6: SPECTROPHOTOMETRY

Introduction
Light and electromagnetic radiation
Spectrophotometry and Spectrophotometer
Absorption Laws
Beer’s Law
Lambert’s Law
Beer-Lambert’s Law
Molar Extinction Coefficient
Transmittance
Visible Light and Ultraviolet Spectroscopy
Instrumentation
Photoelectric colorimeter
UV-Visible Spectrophotometer
Standard or Calibration Curve
Limitation and Deviation of Beer-Lambert Law
Applications of UV-Visible Spectrophotometry

CHAPTER-7: CHROMATOGRAPHIC TECHNIQUES

Introduction
Principle of Chromatography
Types of Chromatography Techniques
Paper Chromatography
Thin Layer Chromatography
Column Chromatography
Adsorption Chromatography
Ion-exchange chromatography
Molecular Size Exclusion Chromatography
Affinity chromatography
Gas Chromatography
High Pressure Liquid Chromatography

CHAPTER-8: IMMUNOLOGICAL TECHNIQUES

Introduction
Structure of antibodies and its types
Antigenicity and Immunogenicity
Generation of Antibody
Production of Polyclonal Antibody
Production of Monoclonal Antibody
Antigen-Antibody Interaction
Techniques Based on Immunology
Immunoprecipitation
Working Principle of Immunoprecipitation
Factors affecting immunoprecipitation
Types of immunoprecipitation
Co-immunoprecipitation
Chromatin immunoprecipitation
RNA immunoprecipitation
Tagged protein immunoprecipitation
Factors affecting immunoprecipitation
Immunodiffusion
Types of immunodiffusion
Single immunodiffusion
Radial immunodiffusion
Double immunodiffusion
Immunoelectrophoresis
Types of immunoelectroporesis
Cross-over immunoelectroporesis
Rocket immunoelectroporesis
Grabar-Williams immunodiffusion
Clarke-Freeman 2-D immunoelectrophoresis
Precipitation Methods in Solution
Agglutination
Types of Agglutination
Agglutination inhibition test
Radioimmunoassay
Enzyme Immunoassays
Enzyme-Linked Immunosorbent Assay
Types of ELISA
Elispot Assay
Western Blotting Technique
Immunofluorescence
Flow Cytometry
Immunohistochemistry
Immunomicroscopy

CHAPTER-9: POLYMERASE CHAIN REACTION

Introduction
The working of PCR Cycle
Kinetics of PCR
Types of PCR
Reverse Transcriptase PCR
Differential Display PCR
Real Time PCR or Quantitative PCR Multiplex PCR
DNA intercalating agents
Inclusion of Molecular Beacon in PCR
Multiplex PCR
Nested PCR
Touchdown PCR
Hot-start PCR
Colony PCR
How to design a good primer?
Applications of PCR
Rolling cycle amplification

CHAPTER-10: PCR MUTAGENESIS AND MICROARRAYS

Introduction
Oligonucleotide-Directed Mutagenesis
PCR-Amplified Oligonucleotide-Directed Mutagenesis
Random Mutagenesis
DNA Microarray Technology
Basic principle of DNA microarray technology
Applications of microarray technology
Protein Arrays

CHAPTER-11: DNA SYNTHESIS AND SEQUENCING

Introduction
Chemical synthesis of oligonucleotides
The phosphoramidite method
Uses of synthesized oligonucleotides
DNA sequencing
Chemical method of DNA sequencing
Enzymatic method of DNA sequencing
Pyrosequencing
Automated DNA sequencing
Large-scale DNA sequencing
Shotgun Method of Whole Genome Sequencing

CHAPTER-12: RECOMBINANT DNA TECHNOLOGY

Introduction
How do we clone a DNA sequence?
Nucleic acid cutting enzymes
Restriction endonucleases and DNA modification
Cutting and joining of DNA
Types of restriction endonucleases
Ligation - Joining of cut DNA fragments
Restriction mapping
How to construct a restriction map
Restriction Fragment Length Polymorphism
Molecular cloning
Other Artificial Vectors
Inserting DNA into Vector
How to detect inserted vector
Transformation and Selection
Constructing DNA libraries
Genomic Library
Complementary DNA (cDNA) Library
Screening Techniques of Cloned DNA

CHAPTER-13: MOLECULAR HYBRIDIZATION TECHNIQUES

Introduction
In-situ Hybridization
Hybridization of DNA and RNA
Southern Hybridization
Northern Hybridization
Zoo Blotting
Genomic in-situ Hybridization
Applications of GISH
Nucleic Acid-Protein Interaction Study
RNase Protection Assay
DNA–Protein Interaction Study
Electrophoretic mobility shift assay
DNA Footprinting
Ribonuclease (RNase) Footprinting
Screening Techniques of Cloned DNA
Colony hybridization
Antisense Technology
Antisense oligonucleotides
RNA interference

CHAPTER-14: TRANSFECTION AND MUTATION DETECTION

TECHNIQUES

Introduction
Types of gene transfer in animal cells
Biological Transfection
 Chemical Transfection
Mutation Detection Techniques
Cytogenetic techniques
Molecular techniques
Detection of Known Mutation
Ligase Chain Reaction
Amplification refractory mutation system
Multiplex ligation-dependent probe amplification
Detection of Unknown Mutations
Single Strand Conformational Polymorphism
Denaturing-high-performance liquid chromatography
Denaturing Gradient Gel Electrophoresis
Heteroduplex analysis of mutation
Restriction fragment length polymorphism
Cleavage Method of Mutation Detection
Mismatch Chemical Cleavage
Protein Method of Mutation Study
Protein Truncation Test

APPENDIX: Solutions and Buffers

LISTS OF SUGGESTED READINGS

INDEX

View publication stats