Restriction digests of the clone give the following sizes (kb): PstI--3.0, 1.1, 1.1; EcoRI--5.2; HindIII--5.2. The clone contains two copies of the insert. The insert contains the following restriction sites (approximate kb from the 5' end): PvuII--0.06, 0.82; SphI--0.35; NcoI--0.38; SmaI--0.79. 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. A hybridization probe may be generated using the following vector specific PCR primers: modified T3 = 5'-CCCCTCACTAAAGGGAACAAAAGCTG-3' and modified T7 = 5'-CGCGTAATACGACTCACTATAGGGCGAA-3'. The suggested PCR generated probe encodes the last 617bp of the aph(3')-IIa gene plus 290bp of the bleomycin resistance gene from Tn5. A better probe for the aph(3')-IIa gene would be the 383bp PstI/NcoI fragment. |
