ABSTRACT
Modern molecular biology begins usually with a catalog, a list of naturally occurring enzymes that have been identified, purified, and in some cases mutated in order to facilitate the manipulation of DNA and RNA molecules. The currency of most cloning experiments is the plasmid, a circular piece of DNA found in bacteria that usually ranges from 2,000 to 14,000 base pairs in length. This chapter illustrates each of the steps in amplifying, purifying, and screening plasmid DNA, and explains design of a ligation experiment with an example that includes key troubleshooting steps. The whole amount of DNA in a ligation reaction is usually transformed into highly competent cells; note that this is about tenfold as much as is used for ordinary plasmid amplification. Traditional DNA purification methods involve ultracentrifugation on a CsCl density gradient. Restriction mapping resulted from the identification and exploitation of the way in which DNA is exchanged and eliminated in nature.
