Restriction digests of the clone give the following sizes (kb): HindIII/XhoI--3.0, 0.4; HindIII--3.4; XhoI--3.4. The insert contains the following restriction sites (approximate kb from the 5' end): ClaI--0.04; ScaI--0.11; SacI--0.32. A single gel purification of the PCR generated probe is necessary since flanking regions will co-amplify with the gene specific sequence. Failure to do so often results in high backgrounds and false positives with clinical E. coli strains. Due to sequence similarity between aac(6')-Ib and aac(6')-IIa genes, the aac(6')-Ib probe is used to detect both genes. A hybridization probe may be generated using the following vector specific PCR primers: modified T3 = 5'-CCCCTCACTAAAGGGAACAAAAGCTG-3' and modified T7 = 5'-CGCGTAATACGACTCACTATAGGGCGAA-3'. |
