Restriction digests of the clone give the following sizes (kb): EcoRV/XhoI--2.9, 1.3; EcoRI--4.3; XhoI--4.3; EcoRI--4.3; HindIII--uncut; BamHI--4.3. The insert contains the following restriction sites (approximate kb from the 5' end): HpaI--0.01; BclI--0.41, 0.79; NdeI--0.46; BstXI--0.53. Two rounds of gel purification of the restriction digest generated probe is necessary. Failure to do so often results in high backgrounds (due to flanking regions in the plasmid insert) and false positives with clinical E. coli strains. The 375bp BclI fragment is recommended as a hybridization probe for aac(2')-Ia. |
