A Practical Approach to Molecular Cloning

This introductory chapter depicts our efforts to cover all the important aspects that a beginner should learn and know to work successfully in a molecular biology laboratory. Familiarity with all kinds of laboratory safety rules, including proper handling of various hazardous chemicals, do’s and don’ts of various procedures, and storage of various chemical, biological, and radiological reagents and the hazards associated with them, is mandatory for every beginner in this area and hence the discussion about these topics is the foremost element, to begin with. Tips for personal protection and safety of the experimenters during the experimentations with these hazardous agents are also mentioned at different places in this chapter. Next, the authors include a virtual walk-through of the laboratory to provide knowledge of the location of the entire laboratory and departmental equipment and their handling. The importance and requirements of mathematical and other experimental skills, starting with cleaning glassware and autoclaving to designing a cloning experiment, are discussed categorically in an elaborate manner that should benefit the beginner experimenter. 

This chapter provides the experimenter with an overview of the microbiological procedures and techniques indispensable for molecular cloning. We presented detailed descriptions of all the essential techniques associated with handling the laboratory’s favorite microorganism, the gram-negative bacteria Escherichia coli. The chapter begins with the step-by-step procedures of different aseptic techniques, description, compositions, and preparation procedures of different microbial growth media, which is followed by exhaustive procedures of inoculating and culturing E. coli in those growth media and methods associated with monitoring their growth. We also discuss different storage methods used to preserve Escherichia coli on long-term and short-term basis that are routinely carried out in cloning procedures. These rules that the experimenter needs to adhere to are outlined to achieve successful results.

This chapter introduces experimenters to the handling and the use of plasmids as cloning vehicles. The chapter begins with the fundamental biology and classifications of different plasmids, followed by various protocols to isolate plasmid DNA from E. coli by alkaline lysis and boiling methods from small, medium, and large cultures. Various critical parameters and notes to be considered while performing each step are also included at different places, which is essential for successful isolation. Finally, purification, storage of plasmid DNA, and recipes for reagents and solutions sum up this chapter.

Isolation and purification of genomic DNA is an important procedure in modern molecular biology. This chapter begins with an introduction to eukaryotic genomic DNA followed by various optimized protocols of their isolation from various sources. The three most commonly used procedures of genomic DNA from bacterial cells, plant tissues, and whole blood in the laboratory are presented in vivid detail. Furthermore, we have included recipes for various reagents and solutions required for the isolation of genomic DNA samples. As usual, the critical parameters and notes to be taken into consideration are also mentioned along with the protocols after the appropriate steps.

Analysis of isolated genomic and plasmid DNA samples is critical and vital to assess their quality and quantity. Here, we have included methods to analyze the plasmid and genomic DNA samples by gel electrophoresis and spectrophotometric methods. The principles and factors affecting both agarose and polyacrylamide gel electrophoresis are discussed. Along with this, protocols are cited for analyzing and recovering DNA from agarose and polyacrylamide gels. The recipe for the buffers and solutions required for each are mentioned for the convenience of beginner experimenters. Spectrophotometric estimation of isolated DNA, included at the end of the chapter, will provide insight into the purity of the DNA sample.

The strategies and methods of construction of recombinant DNA molecules, which is the central objective of this book, are elaborated in this chapter. A specific strategy associated with a particular molecular cloning procedure is extremely crucial for its successful execution. Various cloning strategies and procedures, which are successfully used in different laboratories, are described at the beginning of the chapter. Various steps of cloning are described in detail, including the methods of preparation of the vector and insert DNA samples either by digestion with a restriction enzyme or by polymerase chain reaction, modification of vectors and insert DNA, ligation, and transformation. Moreover, the inclusion of the properties and utilities of various end modification enzymes like Klenow, alkaline phosphatases, etc., used in making both the vector and insert molecules compatible for ligation (essential to create successful recombinant molecules) fulfills the goal of the chapter. The process of cutting and joining, though it may seem simple, can pose problems in reality, leading to unsuccessful ligation for which a rigorous troubleshooting guide is provided. The process of making a strain of bacteria ‘competent’ to uptake the ligated DNA by transformation (protocol included) is also included in this chapter. The buffers and solutions required for the entire process can be found at the end of the chapter.

Identification of correct recombinant clones using an appropriate screening strategy following ligation reaction and transformation constitutes one of the most vital methods in molecular cloning. Introducing the commonly used screening strategies used to identify the correct recombinant clone to a newcomer forms the basis of this chapter. We include various methods to verify true recombinants, beginning with a classic way of initial selection of the transformants on antibiotic-containing medium followed by powerful means of blue-white colony screening strategy, a precise screening strategy using restriction enzyme digestion, and a quick screening strategy using polymerase chain reaction (PCR). The reagents and solutions required for each process can be found at the end of the chapter.