Dntps mix

In order to predict the virulence potential of Salmonella isolates from different sources, the presence of 6 virulence genes (spiA, pagC, msgA, sipB, spaN, and spvB) was screened as two runs; that is, run 1 consisted of 4 (spiA, pagC, msgA, sipB) and run 2 consisted of 2 (spaN and spvB) genes, respectively, using multiplex PCR assays.
The forward and reverse primers used for detection of virulence gene and the amplification conditions were adapted from Versalovic et al. [18 ]. Amplifications were performed in a 25 μL reaction mixture comprising 2.5 μL of template DNA, 16.3 μL of RNase free H₂O, 2.5 μL of 10x PCR buffer, 3.0 μL of 50 mM MgCl₂, 0.15 μL of Taq 5 U/μL [Thermo Fisher], 0.5 μL of 10 mM dNTPs mix (New England Biolabs®), and 0.05 μL of 0.1 mM forward and reverse primers. Amplification was performed in a thermocycler (MJ Research, PTC 200 USA) under the following conditions: 1 cycle of initial denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, and annealing for 1 min at 53°C for spvB, spiA, pagC, and msgA and at 56°C for sipB and spaN and 2 min at 72°C, with a final cycle of 10 min at 72°C, followed by a hold at 4°C. The resulting PCR products were subjected to horizontal gel electrophoresis in 2% agarose and visualized using BioDoc-It™ Imaging (UVP, USA) and images retrieved from the computer in the data office.

Amplification of cDNA obtained was done using gene specific primers designed and listed in Supplementary Table 2. Briefly, 1 μl of cDNA sample was used to prepare a 10 μl reaction mixture containing Taq Pol enzyme (5 U/μl) (NEB), dNTPs mix (0.25 mM) (NEB), Taq Pol enzyme buffer (1X) (NEB), forward and reverse primer (0.2 μM each), and Nuclease free water (Genei). The tubes were placed in Mastercycler pro (with vapo. protect, Eppendorf) for PCR amplification. The obtained amplified PCR products were run on 1.8% agarose gel after mixing with DNA loading dye. Gel electrophoresis was carried out at 90 V, bands visualized and the band intensity measured using Quantity One Software version 4.6.5 (Basic) from BIO-RAD. GAPDH was used as endogenous control.

All chemicals used in this study were of analytical reagent grade. Ampicillin, isopropyl-β-D-1-thiogalactopyranoside (IPTG), phenyl sepharose, sephacryl S-200 HR, oligonucleotides and other chemicals were purchased from Sigma-Aldrich, Co. (St. Louis, USA). Restriction enzymes, DNA ladders, dNTPs mix and other material used for cloning were obtained from New England Biolabs Inc. (MA, USA). Ni-NTA agarose beads, SDS-PAGE molecular weight marker were purchased from GE Healthcare (UK). DNA gel extraction kit was procured from Qiagen Inc.(CA, USA).