Restriction digests of the clone give the following sizes (kb): HindIII--3.5; XhoI--3.5; HindIII/XhoI--3.1, 0.4. The insert contains the following restriction sites (approximate kb from the 5' end): BalI--0.08; RsaI--0.20. 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'. |
