MICB 325 Study Guide - Quiz Guide: Mhc Class I Polypeptide-Related Sequence B, Aeration, Flowchart
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Recall that r. capsulatus cannot be transformed using plasmid dna, and so conjugation will be used to move plasmids into r. capsulatus: pool all lacz- (white) tetr e. coli colonies in liquid medium. With a large number of colonies (~104) we have the entire r. capsulatus genome, as a set of overlapping 3 to 6 kb fragments, each represented by the cells in each white colony. One would probably suspend these cells in about 10 ml of culture medium supplemented with 15% glycerol as cryoprotectant ("antifreeze") and divide it into about ten 1 ml samples (1010 cells per ml). Use conjugation to transfer plasmids from auxotrophic e. coli into a prototrophic, crt- r. capsulatus strain. Mix about 107 cells from the gene library with an equal number of crt- r. capsulatus cells, incubate overnight, then spread mixture on plates of minimal medium containing tetracycline to select for plasmid recipients, and screen for red colonies:
