How is the plasmid DNA purified?
There are many different methods for performing plasmid DNA ‘mini-preps’. In the one described in this protocol, the bacterial cells are pelleted, resuspended in a small volume of buffer, and lysed open with a combination of detergent and alkali to release their contents. The bacterial proteins and chromosomal DNA are precipitated together using a high salt solution. RNA is degraded via an enzyme, RNAse, present in the resuspension buffer. The plasmid DNA is then further purified from proteins and lipids via extraction with the organic solvents phenol and chloroform. The DNA is then precipitated in ethanol (EtOH), resuspended in dH2O, and analyzed via restriction enzyme digestions. NOTE: Instead of the mini-prep protocol described here, we may use a commercially available DNA purification kit. In this case the protocol will differ. How are the plasmid DNAs analyzed? In order to determine whether there are differences between the plasmid DNA present in different colonies, an aliquot of
