Reactions using primers Saka1a-F/Saka2b-R and SG-F/SG-R and SI-F/SI-R and ESSF/ESSR were optimized in a 50 μl reaction mixture consisting of 5 μl of the bacterial genomic DNA ABT-737 clinical trial solution (50 ng), 3 mM MgCl2, 0.25 μM (each) dATP, dCTP, dTTP and dGTP; 2 U Taq DNA polymerase, 1.25 μl (0.25 μM each) primers and 33.1 μl nuclease free water. PCR products were analyzed using 2% (w/v) agarose gel electrophoreses in 0.5 × TBE buffer and a constant voltage of 90 V to confirm the presence of amplified DNA. PCR assays using primers for

zpx and gluA/gluB were according to parameters and conditions reported by the authors who Wortmannin datasheet originally described each PCR assay. For BAM primers (350 bp product), initially the PCR analysis was performed on all of selleck screening library the strains using reaction that used the 62°C annealing temperature. However, eight of the strains produced multiple bands in addition to the 350 bp amplicon. Gradient PCR analysis of these strains was performed to find the best annealing temperature that give only one band (unpublished data). From this analysis, an annealing temperature of 50.5°C was selected to complete the study. Surprisingly, the lower annealing temperature gave one band which upon DNA sequencing appeared to be the correct one while the other non-specific bands disappeared. This unexpected result might be due to the use of the Invitrogen Platinum

PCR super mix that was used at 50.5°C but not at other temperatures. Table 1 Oligonucleotide primer pairs and PCR running conditions used in this study Primer Sequence 5′ to 3′ Targeted site Amplicon size (bp) Reference SG-F GGGTTGTCTGCGAAAGCGAAa ITS-G 282 Liu et al., [44] SG-R GTCTTCGTGCTGCGAGTTTG ITS-G & ITS-IA     SI-F CAGGAGTTGAAGAGGTTTAACTb ITS-IA 251 Liu et al., [44] SI-R GTGCTGCGAGTTTGAGAGACTC ITS-G & ITS-IA     Saka 1a ACAGGGAGCAGCTTGCTGCc

V1g 952 Hassan et al., [45] Saka 2b TCCCGCATCTCTGCAGGA V3h     Zpx F GAAAGCGTATAAGCGCGATTCd zpx 94 Kothary et al., [13] Zpx R GTTCCAGAAGGCGTTCTGGT       BAM122 AWATCTATGACGCGCAGAACCGe zpx 350 Kothary et al., [13] BAM123 AAAATAGATAAGCCCGGCTTCG       EsgluAf TGAAAGCAATCGACAAGAAGf gluA 1680 Lehner et al., [3] EsgluAr ACTCATTACCCCTCCTGATG       EsgluBf TGAGTGAAGCACCGACGCAGf gluB 1720 Lehner et al., [47] EsgluBr GTTACGTCACAGGTTTTGAT       ESSF GGATTTAACCGTGAACTTTTCCi Celecoxib ompA 469 Nair and Venkitanarayanan [46] ESSR CGCCAGCGATGTTAGAAGA       a&b Running conditions; 94°C for 10 min; 30 cycles of 94°C for 30 sec each; 57°C for 1 min; 72°C for 1 min; a final extension period of 5 min at 72°C. c Running conditions; 95°C for 4 min; 30 cycles of 95°C for 60 sec each; 50°C for 1 min; 72°C for 90 sec; final extension period of 4 min at 72°C. d&e Running conditions; The hot start polymerase was activated by incubation for 15 min at 95°C; followed by 35 cycles of 1 min at 95°C; 62°C for zpx primers (50.