Restriction digests of the clone give the following sizes (kb): EcoRI/HindIII--3.0, 0.5; EcoRI--3.5; HindIII--3.5. 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'. |
