Restriction digests of the clone give the following sizes (kb): HindIII--4.6; BamHI--4.6; Sau3AI--1.0, 0.7, smaller fragments; KpnI--3.9, 0.7; XbaI/HindIII-- 3.0, 1.6. The insert contains the following restriction sites (approximate kb from the 5' end): ClaI--0.67; SacII--0.80; Asp718I--0.93; KpnI--0.93; NcoI--1.01; EcoRV--1.41. 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 485bp Asp718I/EcoRV fragment is recommended as a hybridization probe for aac(3)-IIb. |
