Finally my book has published from Kantipur College of Medical Science, Sitapaila, Kathmandu.
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Contact:
Kiran Babu Tiwari (Author): kbtiwaree@gmail.com, 00977-9841-374738
College: 00977-1-4279723
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Preface:
Working for years in Central Department of Microbiology, I felt the necessity of a practical hand book on Microbial Genetics and Molecular Biology as a text book with the practical specially designed towards the M. Sc. Microbiology course of Tribhuvan University, Nepal. The aim of this book is to provide basic and advanced practical exercises in microbial genetics and molecular biology useful for masters’ level students and a guideline material for the tutors as well.
While writing this manual, I considered our limited resources and the need to develop experiments that can illustrate and develop concepts in microbial genetics and molecular biology. So the experiments which have been included in the hand book could be carried out in our country context as well.
The hand book explains experiment in such a way that students can understand objectives and principles before starting the procedure. Procedural sections are also written to make the students and tutor/s understand the sequential flow of steps to be carried out. We tried to start from the very beginning: 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. Some of the useful information is also attached under annexes. This is one of the efforts to make students and tutors easy in designing and performing experiments. We hope this book will equally be useful to other disciplines of biomedical sciences. Any comments, suggestions for improving this book/manual will be highly appreciated.
I am indebted to Prof. Dr. Shital Raj Basnyat for writing forewords for this book.
While writing this manual, I considered our limited resources and the need to develop experiments that can illustrate and develop concepts in microbial genetics and molecular biology. So the experiments which have been included in the hand book could be carried out in our country context as well.
The hand book explains experiment in such a way that students can understand objectives and principles before starting the procedure. Procedural sections are also written to make the students and tutor/s understand the sequential flow of steps to be carried out. We tried to start from the very beginning: 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. Some of the useful information is also attached under annexes. This is one of the efforts to make students and tutors easy in designing and performing experiments. We hope this book will equally be useful to other disciplines of biomedical sciences. Any comments, suggestions for improving this book/manual will be highly appreciated.
I am indebted to Prof. Dr. Shital Raj Basnyat for writing forewords for this book.
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Contents:
CONTENTS
Orientation: Orientation of Microbial genetics / Molecular Biology Laboratory.................1
Principles: Principle of nucleic acids isolation from bacteria .............................................3
Experiment 1: Calibration of micropipettes ..................5
Experiment 2: Preparation of Tris buffers: Tris-HCl, Tris-EDTA and TAE ....................................7
Experiment 3: Isolation of DNA from human blood......10
Experiment 4a: Extraction of chromosomal DNA from Gram positive bacteria .......................12
Experiment 4b: Extraction of chromosomal DNA from Gram negative bacteria ......................14
Experiment 4c: DNA extraction from yeasts ................16
Experiment 4d: Isolation of nucleic acid (DNA / RNA) from Coliphages .......................................18
Experiment 5a: Extraction of Plasmid DNA from bacteria .......................................................20
Experiment 5b: Isolation and purification of bacterial plasmids using Qiagen spin Miniprep system .............................................22
Experiment 6a: Extraction of RNA from yeasts .............30
Experiment 6b: Extraction of RNA from bacteria ..........32
Experiment 7: Spectrophotometric analysis of DNA in extracted solution ..............................34
Experiment 8: Agarose gel electrophoresis of DNA.......36
Experiment 9a: Demonstration of induction/repression mode of lac-operon regulation system in Escherichia coli..................................40
Experiment 9b: Determination of level of induction of lac-operon in Escherichia coli....................42
Experiment 10: Conjugation in bacteria: Co-culture of MDR Salmonella enteritidis Typhi with Escherichia coli HB101 and detection of transconjugants by antibiotic selection method.............................................44
Experiment 11: Transformation of Escherichia coli HB101 by pUC18 or pBR322..........................46
Experiment 12: Study of performance of HindIII and EcoRI Restriction enzymes stored in lab.........49
Experiment 13a: Construction of recombinant DNA: Preparation and purification of insert (Lambda DNA fragments) and vector (pUC18) DNA ....................................54
Experiment 13b: Construction of recombinant DNA: Ligation of purified insert (Lambda DNA fragments) and vector (pUC18) DNA.....57
Experiment 14a: Amplification of recombinant DNA: Transformation of recombinant pUC18 DNA in Escherichia coli HB101 .............59
Experiment 14b: Detection of recombinant DNA: Extraction of recombinant pUC18 DNA from transformed Escherichia coli HB101 clones .......................................................61
Experiment 14c: Detection of recombinant DNA: Detection of recombinant pUC18 DNA in plasmid extracts of transformed Escherichia coli HB101 clones by agarose gel electrophoresis .................................63
Experiment 15a: Amplification of Escherichia coli DNA (genes) by Polymerase Chain Reaction (PCR) ................................................65
Experiment 15b: Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction (RAPD-PCR) .......................................................68
Experiment 16: Bacterial plasmid profiling....................71
Experiment 17: Construction of simple restriction map of a plasmid DNA .....................................73
ANNEXES
Annex I: Molecular Biology components ............76
Annex IIa: Preparation of buffers.........................78
Annex IIb: Tri buffers ........................................81
Annex III: Phenol .............................................83
Annex IV: Bacterial nucleoid staining ..................85
Annex V: Antibiotic susceptibility testing (AST) of bacteria ...........................................86
Annex VI: Broth dilution method to determine Minimum inhibitory concentration (MIC) .......................................................88
Annes VII: Generation of actinomycete mutants using sodium azide and their analysis..........89
Annex VIII: Replica plating ..................................91
Annex IX: Ames test .........................................94
Annex X: Determination of ciprofloxacin related mutation rate in wild type Salmonella typhi................................................98
Annex XI: Marker - 1 Kb ladder DNA..................100
Annex XIIa: Escherichia coli host strains for transformation ................................102
Annex XIIb: Alpha-Complementation ...................106
Annex XIIIa: Construction of simple restriction map of pBR322 using PstI, HindIII and EcoRI ......................................................107
Annex XIIIb: pUC vectors ....................................111
Annex XIIIc: pBR322 vector ...............................113
Annex XIV: Star activity of Restriction enzymes ....114
Annex XV: PCR calculations ..............................117
Annex XVI: Southern Blotting ............................120
Annex XVII: Ethidium bromide ............................123
Annex XVIII: Molarity of HCl ................................128
Bibliography: Bibliography ....................................129
