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Rapid Verification Of Terminators Using The Pgr-Blue Plasmid And Golden Gate Assembly, Jace C. Bradshaw, Allea Belle Gongola, Nathan S. Reyna
Rapid Verification Of Terminators Using The Pgr-Blue Plasmid And Golden Gate Assembly, Jace C. Bradshaw, Allea Belle Gongola, Nathan S. Reyna
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The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony …