Genomic Insertion Protocol

Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.