Plasmid DNA was extracted from N315 cells (bearing the pN315 plasmid) cultured in 5.0 ml brain–heart infusion broth and purified by the Plasmid Mini kit (Qiagen, Tokyo, Japan). The average yield of DNA appeared to be ~50 ng. To confirm that the extracts contained the plasmid bearing the ß-lactamase gene, they were subjected to PCR amplification using the primer set K. Agarose gel Temsirolimus mouse electrophoresis clearly showed a single distinct large band corresponding to the size of the expected PCR product (similar to the result
in Figure 2, Ref. N315). Attempts have been made to extract the plasmid DNA from BIVR cells, such as K744 and five other strains, but the yield was consistently undetectable except for the K2480 cells, which showed a trace amount of DNA. PCR amplification of blaZ taking the K2840 extracts as the template yielded JNJ-26481585 no visible band. The BIVR cells, K744 and K2480, were transformed with plasmid
DNA extracted from N315 cells. Selection of the transformants for ß-lactam resistance was difficult because the recipient cells were ß-lactam-resistant beforehand to a certain extent. Thus, transformants were selected on agar plates impregnated with a 1.5-fold MIC equivalent of ampicillin and obtained from K744 and K2480 strains (K744-T and K2480-T, respectively). Presence of the blaZ gene in the K744-T and K2480-T cells was confirmed selleck chemicals by PCR using whole-cell extracts as the template, and subsequent agarose gel electrophoresis yielded a single DNA band corresponding
to that obtained from N315 cells (Figure 3). Note that the amount of PCR products using K744-T and K2480-T DNA as the template appeared low compared with that from N315 cells (Figure 3). The identity of untransformed and transformed cells was confirmed by pulse-field gel electrophoresis of the chromosomal DNA treated with SmaI. Unsuccessful attempts were made to transform FDA209P with the pN315 plasmid. The reasons for failure of this transformation experiment remain obscure. Figure 3 PCR products of the blaZ gene. The primer sets in alphabetical order correspond with that in Table 2. Agarose Calpain gel electrophoresis was carried out as described in the legend to Figure 2. Only a part of the electrophoretogram is shown. Arrow and bp, the amplicon size; N315, K744-T and K2480-T were the source of the template DNA. ß-lactamase activity was determined using N315, K744-T and K2480-T cells. The results showed that activity in N315 cells appeared to be 0.74 U, while the levels in K744-T and K2480-T cells were undetectable (Table 2). Plasmid DNA from K744-T was undetectable, but a trace amount was extracted from K2480-T comparable with the level from the untransformed parent cells. Attempts have been made to amplify the blaZ DNA using the column eluate of the extracts as the template.