Of late, genetic engineering or recombinant DNA technology has been emerged as a very potential filed in molecular biotechnology. DNA isolation and characterization are the requisites of many fundamental molecular researches and thus have become an integrated part in the study of both basic and applied molecular biology. In the basic fields, researches are carried out to know the functions of different genes, their expression patterns etc. which in turn, is making pace for the advanced and applied researches. In the applied fields, molecular techniques have been adopted to produce thousands of commercial products including different agricultural and pharmaceutical products that are being served to fulfill regular consumer requirements. Therefore, recombinant DNA technology is playing very important role in both basic and applied researches. In this chapter, basic and fundamental techniques as well as recent advanced techniques used in molecular biotechnology have been described along with some diagrammatic illustrations. The process for isolation and characterization of a gene has been described sequentially and step-wise to make it easily understandable. In addition, the entire process for cloning a gene in different circumstances has been presented in schematic-form in the last part of the chapter for a comprehensive review.