Sephadex g 50 fine resin

Fifty micrograms of each plasmid was digested using BsaI (New England Biolabs) to exclude the nonviral sequences at the 3′-end. RNA was then synthesized from a template of the linearized DNA using T7 polymerase and purified with a gel filtration column (HiLoad 16/60 Superdex 75, or 200) (the purity of RNAs; at least 90%) as described by Mckenna et al. (21 (link)) and Shimoike et al. (22 (link)).
The 5′-end ³²P-labeled template RNAs were prepared as follows: the calf intestinal alkaline phosphatase (Takara) was used to dephosphorylate the 5′-end of the purified template RNAs. Subsequently, the ³²Phosphate was bound to the 5′-end of the dephosphorylated template RNA by the T4 polynucleotide kinase (Takara), followed by phenol/chloroform, and precipitated by ethanol. The unreacted γ-32P-ATP in the RNA was then removed using Sephadex G-50 fine resin (GE Healthcare). The ³²P-labeling efficiencies of RNAs were found to be 49% to 51%.

Genomic DNA was isolated using the InstaGene Matrix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. All primer sequences for amplification and sequencing were obtained from the MLST Databases at the University of Warwick (www.mlst.warwick.ac.uk/mlst/dbs/Senterica). The PCR cycling conditions were as indicated in instructions posted on the website. PCR products were purified with Sephadex G-50 fine resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Nucleotide cycle-sequencing was performed directly on purified PCR templates using automated Sanger dideoxychain termination methods and the primers described on the MLST website. Sequences of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were compared with the available MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Senterica) to get the allele number and sequence typing (ST) number for each isolate. Sequence information for newly assigned alleles and STs was deposited in the MLST database [14 (link)].

Genomic DNA was isolated using the InstaGene Matrix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. All primer sequences for amplification and sequencing were obtained from the MLST Databases of the University of Warwick (www.mlst.warwick.ac.uk/mlst/dbs/Senterica, accessed on 21 June 2023). The PCR cycling conditions were as indicated in instructions posted on the website. The PCR products were purified using Sephadex G-50 fine resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Nucleotide cycle-sequencing was performed directly on purified PCR templates using automated Sanger dideoxy chain termination methods and the primers described on the MLST website. Sequences of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were compared with those in the MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Senterica accessed on 21 June 2023) to obtain the allele number and sequence type (ST) number for each isolate. Sequence information for newly assigned alleles and STs was deposited in the MLST database [17 (link)].