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In this century, students of biology are confronted with an entirely different scenario. All aspect of biology become more molecular-molecular biology. The tools have transformed our information management, taking access information to new heights. The advances made by the molecular biology tools have been very phenomenal in understanding and solving many of age old problems involved with many plant and animal genomes. These tools have been very dynamic when combined with traditional paths of research to know the structure and functions of millions of genes. The present book chapters contain first hands-on information on methods and protocols in a simplified manner which is very easy to learn and perform. Further, methods and protocols constitute a gold-standard reference for today's scientists who wish to develop and hone their molecular biology skills towards the discovery of new biological relationships. This book has been divided into 10 chapters with each chapter containing introduction, principle, protocol, applications and troubleshooting and it has been written keeping in mind the requirements of graduate/postgraduate students and research scholars

0 Start Pages

Preface   Advances in Biotechnological tools have helped in more unified understanding of many plant and animal genomes and most of the experiments which were unimaginable in earlier years have been performed very efficiently. Most of the experiments involved in molecular biology are difficult to analyze, understand and perform for the beginners. The basic information about techniques in molecular biology are covered in many introductory molecular biology and biotechnology books but protocols of such experiments have been mentioned in very few book chapters. Finding these protocols is very cumbersome for students and researchers who are at the beginning stages of their experiment/research and theoretically any student can learn about any techniques but performing the same at laboratory level is very difficult without first hand experience. Just by learning protocols one cannot do molecular biology experiments and the principle behind becomes very crucial for the effective solution to a particular biological problem. This lab manual gives information on the protocols, principles and applications behind mostly performed experiments and it has been written keeping in mind the requirements of graduate/postgraduate students and research scholars. With the boom in biotechnology research in India many new molecular biology laboratories have been established in many Universities and Research institutes. There has been steep increase in the number of students studying molecular biology and biotechnology courses all over India. The present book chapters contain information on basic protocols in simplified manner which is very easy to learn and perform. This book further gives first hands-on information and it has been divided into 10 chapters with each chapter containing introduction, principle, protocol, applications and troubleshooting. Manuscript of the book contains many protocols and figures which were collected from various websites, research papers and training manuals, we acknowledge all those individuals involved directly or indirectly in preparation of this Laboratory Manual. Most of the existing protocols have been mentioned in a simplified manner just to aid easy understanding for students and helping the same to perform very effectively.

1 Laboratory Equipments and Its Usage

Molecular biology labo-ratories are becoming very common in every University and research institutes and a good laboratory manners is extremely important as many of the equipments are very expensive and sensitive. Hence familiarity with equipments becomes very crucial before starting any experiment.

1 - 8 (8 Pages)
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2 Buffers

Introduction This section of the manual contains instructions for preparing stock solutions that are commonly used in laboratory protocols for the analysis of DNA, RNA and proteins and visualization of gene or protein expression. Each of these stock solutions is provided as either the most convenient concentration or the most frequently used stock concentrations to be used in the protocols of an individual laboratory. All chemicals must be reagent grade or molecular biology grade, and the water used in the preparation of all solutions must be the highest quality available. Use sterile-glass distilled deionised water whenever possible. Unless otherwise stated, most solutions required sterilization either by filtration or autoclaving.

9 - 14 (6 Pages)
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3 Cell Culture

Plant cell culture Introduction As the human population is expanding there has been increasing concern over whether current rates of food productions can support the growing population. In response to the increasing demand, scientists and farmers have been working hard to come up with new ideas to meet these needs. These ideas range from simple procedures including selection of the best seeds to more complex procedures requiring the transplantation of genes into different plant species. This chapter focuses on one of the important aspects of plant science which is the growth of plant cells in an artificial media. This process is known as plant cell culture and can be applied in many different ways. 

15 - 46 (32 Pages)
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4 Nucleic Acid Extraction

DNA Isolation Protocol Introduction Most of the all procedures involved in molecular biology experiments require the purified form of nucleic acid. It forms a fundamental requirements for genome characterization, gene mapping and for identification and isolation of gene for genetic engineering. The most important step involved in removal of proteins which is carried out in different ways. The following protocol is one of the most commonly used protocols in animal, plant and microbial samples.

47 - 64 (18 Pages)
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5 Determination of Quality and Quantity of Nucleic Acids

Introduction In order to use a fine quality of DNA for various molecular biological work like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random primer labeling), etc., quality and quantity of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.

65 - 70 (6 Pages)
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6 Polymerase Chain Reaction

Introduction This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).

71 - 84 (14 Pages)
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7 Electrophoresis

Introduction Gel electrophoresis is a technique for separating charged molecules with different sizes. Two kinds of gels are commonly used: agarose and polyacrylamide. Agarose gels can be applied to a wider range of sizes than polyacrylamide gels.  By using standard agarose electrophoresis, nuclei acids up to 50 kb may be separated. If Pulsed Field Gel Electrophoresis is used, the upper limit can be extended to 10 Mb. Polyacrylamide gels may separate nucleic acids that differ in length by only 1 nucleotide if their length is less than 500 bp (Sambrook et al., 1989).

85 - 100 (16 Pages)
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8 Restriction Digestion of Enzymes

Introduction Restriction enzymes, recognizes specific base sequences in double-helical DNA and cleaves at specific places, both strands of a duplex containing the recognized sequences. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned. Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s.

101 - 108 (8 Pages)
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9 Blotting

Southern Blotting Introduction Southern hybridization was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. It is also called Southern blotting since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting. In Southern hybridization, a sample of DNA containing fragments of different sizes is subjected to electrophoresis using either polyacrylamide or agarose gel.

109 - 132 (24 Pages)
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10 Cloning

Introduction Only after the discovery of restriction endonucleases it became possible to digest any DNA molecule at a specific site and any two pieces of DNA could be joined together with the help of another modifying enzyme, the DNA ligase. Consequently, a new branch of molecular biology, namely genetic engineering has been developed. With the help of genetic engineering techniques, it is now possible to identify and isolate the gene (for a protein of interest and insert it into the genome of another organism with the help of a suitable vehicle. The foreign gene thus cloned, can be amplified to produce millions of its copies. The insert gene can be expressed by the host to synthesize the gene product. The technique can also be used for understanding the regulation of expression of the cloned gene. The possibilities are endless and probably sky is the only limit. In this chapter we will briefly discuss the basic principles and protocol involved in gene cloning.

133 - 144 (12 Pages)
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11 Sequencing of Nucleic Acids

Introduction DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. The most commonly used sequencing method is the dideoxy method. This method uses dideoxynucleotide triphosphates (ddNTPs) which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent (Sanger et al., 1977).

145 - 158 (14 Pages)
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12 End Pages

Index A Agarose 5, 6, 7, 63,66, 74, 78, 85, 86, 87, 88, 89, 103, 105, 109, 110, 112, 117, 120, 123, 125, 126, 156 Agarose electrophoresis 85, 100 Agarose gel electrophoresis 78, 86 Agro bacterium-mediated transformation 135 Amniocentesis 42 Amplify DNA for cloning 76 Anchored PCR 79 Archaeology 77 B Biolistic/gene gun transformation protocol for RIC 138 Buffer 9, 10, 11, 13, 14, 21, 29, 40, 48, 50, 54, 55, 56, 61, 67, 71, 72, 73, 79, 82, 87, 89, 90, 104, 106, 107, 113, 114, 117, 119, 120, 123, 124, 126, 130, 131, 137, 140, 148, 151, 152 C Cancer cells 41 Cancer research 41 Cell membrane 47 Cell suspension 17, 23, 34, 43 Cell-based DNA cloning 77, 135 Cell-based manufacturing 41 Chelating agent 47 Cloning verification 106 Cultured cells 40, 41, 42

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