Working for years, I ever felt the necessity of a practical book on Microbial Genetics and Molecular Biology as a text manual for M. Sc. Microbiology students who are studying in the Central Department of Microbiology and other colleges under or affiliated to Tribhuvan University, Kathmandu, Nepal. The aim of this book is to provide them the basic and advanced practical knowledge in the field.
While writing this manual, I considered our limited resources and facilities, so that experiments that illustrate and develop concepts could be conducted.
The manual explains experiments in such a way that students can understand objectives and principles before procedure. Procedural sections are also written to make the students and tutor/s have a sequential flow of jobs. I tried to maintain gradient of difficulties right from micropipette calibration through DNA / RNA extraction, analysis of analyte, qualitative and quantitative determination of lac-operon, conjugation, transformation, DNA cloning, PCR to Restriction mapping. Many useful information / chapters are attached under annexes.
Author
Kiran Babu Tiwari
Central Department of Microbiology
CONTENTS
Orientation: Orientation of Microbial genetics / Molecular Biology Laboratory
Principles: Principle of nucleic acids isolation from bacteria
Experiment 1: Calibration of micropipettes
Experiment 2: Preparation of different Tris buffers: Tris-HCl, Tris-EDTA and TAE
Experiment 3: Isolation of DNA from human blood
Experiment 4a: Extraction of chromosomal DNA from Gram positive bacteria
Experiment 4b: Extraction of chromosomal DNA from Gram negative bacteria
Experiment 4c: DNA extraction from yeasts
Experiment 4d: Isolation of nucleic acid (DNA / RNA) from Coliphages
Experiment 5a: Extraction of Plasmid DNA from bacteria
Experiment 5b: Isolation and purification of bacterial plasmids with Qiagen spin Miniprep system
Experiment 6a: RNA extraction from yeasts
Experiment 6b: RNA extraction from bacteria
Experiment 7: Spectrophotometric analysis of DNA in extracted solution
Experiment 8: Agarose gel electrophoresis of DNA
Experiment 9a: Demonstration of induction/repression mode of lac-operon regulation system in Escherichia coli
Experiment 9b: Determination of level of induction of lac-operon in Escherichia coli
Experiment 10: Conjugation in bacteria: Co-culture of MDR Salmonella enteritidis Typhi with Escherichia coli HB101 and detection of transconjugants by antibiotic selection method
Experiment 11: Transformation of Escherichia coli HB101 by pUC18 or pBR322
Experiment 12: Study of performance of HindIII and EcoRI Restriction enzymes stored in lab
Experiment 13a: Construction of recombinant DNA: Preparation and purification of insert (Lambda DNA fragments) and vector (pUC18) DNA
Experiment 13b: Construction of recombinant DNA: Ligation of purified insert (Lambda DNA fragments) and vector (pUC18) DNA
Experiment 14a: Amplification of recombinant DNA: Transformation of recombinant pUC18 DNA in Escherichia coli HB101
Experiment 14b: Detection of recombinant DNA: Extraction of recombinant pUC18 DNA from transformed Escherichia coli HB101 clones
Experiment 14c: Detection of recombinant DNA: Detection of recombinant pUC18 DNA in plasmid extracts of transformed Escherichia coli HB101 clones by agarose gel electrophoresis
Experiment 15a: Amplification of Escherichia coli DNA (genes) by Polymerase Chain Reaction (PCR)
Experiment 15b: Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction (RAPD-PCR)
Experiment 16: Bacterial plasmid profiling
Experiment 17: Construction of simple restriction map of plasmid DNA
ANNEXES
Annex I: Molecular Biology components
Annex IIa: Preparation of buffers
Annex IIb: Tri buffers
Annex III: Phenol
Annex IV: Bacterial nucleoid staining
Annex V: Antibiotic susceptibility testing (AST) of bacteria
Annex VI: Broth dilution method to determine Minimum inhibitory concentration (MIC)
Annes VII: Generation of actinomycete mutants using sodium azide and their analysis
Annex VIII: Replica plating
Annex IX: Ames test
Annex X: Determination of ciprofloxacin related mutation rate in wild type Salmonella typhi
Annex XI: 1 Kb ladder DNA
Annex XIIa: Escherichia coli host strains for transformation
Annex XIIb: Alpha-Complementation
Annex XIIIa: Construction of simple restriction map of pBR322 using PstI, HindIII and EcoRI
Annex XIIIb: pUC vectors
Annex XIIIc: pBR322 vector
Annex XIV: Star activity of Restriction enzymes
Annex XV: PCR calculations
Annex XVI: Southern Blotting
Annex XVII: Ethidium bromide
Annex XVIII: Molarity of HCl
Bibliography: Bibliography
