Computational protocol: Variations in motility and biofilm formation of Salmonella enterica serovar Typhi

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Protocol publication

[…] Flagella-associated genes, fliC and flgK were selected to confirm the motility in all 60 S. Typhi strains. Oligonucleotide primers specific for each target gene were selected using PrimerSelect (DNASTAR; Lasergene, Madison, WI). Selected primer pairs were then tested using in silico with the PCR amplification program (http://insilico.ehu.es/.25). The sequences of the selected primer used were fliC (5'-AAT CAA CAA CAA CCT GCA GCG- 3') and (5'-GCA TAG CCA CCA TCA ATA ACC-3'); flgK (5'- CAA CAA TTA CGC GAA GCA GA -3') and (3'- TAT AAT CCG TCG CCT GAA CC -5') with amplicon size 704 bp and 584 bp, respectively. The PCR amplifications were carried out in a Master cycler (Eppendorf, USA). The PCR mixture in a total volume of 25 μl contained 50 ng of genomic DNA template, 1X PCR buffer, 2 mM MgCl2, 200 μM of each dNTP, 0.3 μM of each primer, and 0.5U of Taq DNA polymerase (Promega, USA). The cycling con ditions were set at 95°C for 5 minutes (1 cycle), 95°C for 30 s, 55°C for 30 s, 72°C for 1 minute (30 cycles), and 72°C for 8 minutes (1 cycle). The products were then analysed on 1.5% (w/v) agarose gel and run at 100 V for 25 minutes and stained in GelRed. Gel images were captured and analysed using Gel Doc XR (Bio Rad, USA). Selected amplified DNA products were verified by DNA sequencing. […]

Pipeline specifications

Software tools PrimerSelect, DNASTAR Lasergene
Applications Sanger sequencing, qPCR
Organisms Salmonella enterica subsp. enterica serovar Typhi, Homo sapiens
Diseases Infection