Restriction digests of the clone give the following sizes (kb): HindIII--3.0; PstI--3.0; HindIII/PstI--2.9, 0.2. The insert contains the following restriction sites (approximate kb from the 5' end): ClaI--0.01; PvuII--0.18 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'. |
