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The importance of plasmids for molecular research cannot be underestimated. These double-stranded
DNA
units that replicate independently of the chromosomal DNA are as valuable to bacterial geneticists as a carpenter’s hammer. Fortunately, today the mycobacterial research community has a number of these genetic tools at its disposal, and the development of these tools has greatly accelerated the study of mycobacterial pathogens. However, working with mycobacterial cloning plasmids is still not always as straightforward as working with
Escherichia coli
plasmids, and therefore a number of precautions and potential pitfalls will be discussed in this chapter.