Colony PCR Amplification of 7kb Plasmid from Deinococcus aquaticus and cmr from E. coli's pRAD1 Plasmid
Introduction This semester, Leilani Boren, Micheal Dockham and I are transforming the 7kb plasmid from Deinococcus aquaticus with E.coli's chloramphenicol resistance gene via PCR amplification and Gibson assembly. Once assembled, the 7kb plasmid will be farmed and reintroduced into D. aquaticus. To do this, we must first prime D.aquaticus' 7kb plasmid as our vector and E. coli's chloramphenicol resistance gene as our antibiotic resistance gene via PCR. DNA cloning works by inserting a target gene (cmr) into a plasmid (7kb) . Afterward, the plasmid is introduced into bacteria (E.coli) via transformation. Colonies will be screened via selective media (LB + chloramphenicol) and, once verified, will be used to farm recombinant plasmid DNA. Typically, Plasmid DNA is manipulated via restriction enzymes which will cut at cloning sites to create an area for our fragment to be introduced and hybridized. However, we will be using the Gibson Assembly due to successes in combining