The Amplification And Purification Of The Adult Chicken B-Globin Locus Inserted In pB1BR15 Through Culturing Of Transformed E. Coli.
The plasmid pB1BR15, a recombinant, circular DNA duplex containing the adult chicken B-globin locus is the plasmid vector under study. A transformed colony of E. coli, containing this plasmid was cloned and cultured. The general procedure began with the cell culturing phase in order to achieve a maximum cell and plasmid count, to attain a high copy number. The next step was to lyse the cells and separate all of the DNA from the cellular debris. Then the plasmid DNA was separated from the other forms of DNA. The plasmids were observed using an agarose gel where the DNA could be confirmed as that of plasmids. Finally, the plasmids were restricted, run on a gel and then electroeluted. Electroelution was aided by the use of ethidium bromide intercalation and observation with UV light. A special multi-restriction process was performed to arrive at absolute confirmation of the plasmid. Through a series of biochemical techniques this plasmid was amplified, purified and restricted with type II restriction endonucleases.