反应详情

Eight co-integrants each from the crosses summarized in Table 2 were streaked for single colonies (from cultures cryo-preserved in 10% glycerol; described in Section 6.4)) on Luria broth plates with ampicillin (100 μg/ml), spectinomycin (50 μg/ml) and gentamycin (10 μg/ml). Single colonies were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking. Ten microliters (each) from these cultures were spread on a MacConkey Agar (base; Becton Dickson, Difco Division, Franklin Lakes, N.J.) plated with 2% galactose and 100 μg/ml ampicillin. Following overnight growth three types of galactose-resistant colonies appeared on the agar surface: red colonies (with various morphologies), white opaque colonies and white translucent colonies, in numbers varying from a few dozen to several hundred. Resolved co-integrants were among the white translucent colonies, and a single white colony was picked from each spot, and re-streaked for purification on the same MacConkey agar. These purified white colonies were tested for spectinomycin resistance (50 μg/ml) and gentamycin resistance (10 μg/ml). The resultant colony types are summarized below in Table 4 (Gent=gentamycin, Spec=spectinomycin, R=resistance, and S=sensitivity)

1. customwere inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking
2. customre-streaked for purification on the same MacConkey agar