Plasmids were originally isolated from simple organisms such as bacteria, algae or yeasts. They are usually much smaller thanHuman Computer Interaction chromosomes (1 000 to 20 000bp, while chromosomes have millions of base pairs). Plasmids are extrachromosomal circular DNA molecules that replicate autonomously, using their own origins of replication (ori) and encode their own RNA s and proteins. They often carry genes encoding resistance to one or more antibiotics, and can confer this resistance to their bacterial hosts. Molecular biologists have adapted plasmids to serve as vectors to carry foreign DNA fragments that have been inserted (cloned) into them. pUC19 is a popular vector which was derived from a naturally occurring E. coli plasmid that was modified for cloning. The important features are its relatively small size (2,686 bp), a 54 bp polylinker with 21 restriction enzyme recognition sites (the Multiple Cloning Site or MCS), and a short segment of DNA containing the promoter region and first part of the coding sequence for the Lacz (B-galactosidase) gene, that allows for “blue-white” screening for cloned inserts. Figure 3-1. Map of the pUC19 plasmid. Glick&Pastemak. Amp gene PUC19 lacz’ gene Multiple cloning sequence lacl gene Origin of replication The miniprep procedure is used to isolate small plasmid DNA from small volume cultures of bacteria while limiting contaminating proteins and genomic DNA. All plasmid purification protocols take advantage of the relatively small size and covalently closed structure of plasmid DNA. Although there is always some

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