Axyprep plasmid midiprep kit

Chromosomal DNA was isolated by phenol-chloroform extraction. Plasmid DNA was isolated and purified using AxyPrep Plasmid Miniprep Kit or AxyPrep Plasmid Midiprep Kit (Axygen Biosciences, USA) according to the manufacturer's instructions. PCR was carried out with Taq polymerase with initial denaturation of 2 min at 94°C followed by 30 cycles of amplification steps of 30 sec at 94°C, 1 min at 52°C, and 1 min at 72°C. PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences). DNA sequencing was performed employing ABI 3100 automated DNA sequencer using the Big-Dye Terminator Kit (Applied Biosystems, USA).

Routine DNA techniques were done according to [31 ]. Chromosomal DNA was isolated with DNA Express Kit (Liteh, Russia). Plasmid DNA was purified using the AxyPrep Plasmid Midiprep Kit (Axygen Biosciences, USA). PCR was carried out as described [14 (link)] using the primers listed in the Table 1. PCR with primers BgrF and BgrR was carried out with initial denaturation of 2 min at 94°C followed by 10 cycles of first step amplification (40 sec at 94°C, 50 sec at 35°C, and 90 sec at 72°C) and 30 cycles of second step amplification (40 sec at 94°C, 50 sec at 55°C, and 90 sec at 72°C). PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences). Sequencing of PCR products was performed by GenomeLab GeXP Genetic Analysis System using the GenomeLab Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter, USA) and the primers listed in Table 1. Transformation of E. coli and S. agalactiae strains with recombinant plasmid was performed by Gene Pulser Xcell (Bio-Rad Laboratories, USA) as recommended by the manufacturer.