Genomiphi hy dna amplification kit

The whole genome of each filament segment was amplified using the illustra GenomiPhi V2 DNA Amplification kit (GE Healthcare Life Sciences, Pittsburgh, PA) for multiple displacement amplification (MDA; Spits et al., 2006 (link)). Each filament segment was placed into 2.5 μL drops of sample buffer in a petri dish, manually disrupted with sterile metal needles, and pipetted into sterile 0.2 mL microcentrifuge tubes. Each buffer-cell-mix was boiled at 95°C for 3 min, amended by 0.2 μL reaction buffer and 0.5 μL V2 enzyme mix, and incubated at 30°C for 2 h before the reaction was terminated at 65°C for 10 min. The success of the six amplifications was visualized on a 0.8% agarose gel, and the purity of the amplicons were tested by 16S rRNA gene sequencing and phylogenetic analysis with the Silva database release 102 (Pruesse et al., 2007 (link); Quast et al., 2013 (link)) in the ARB software package (Ludwig et al., 2004 (link)). The initial MDA products were reamplified with the illustra GenomiPhi HY DNA Amplification kit (GE Healthcare Life Sciences, Pittsburgh, PA), using 0.5 μL of the 1:10 diluted initial amplicons in a 10 μL final reaction volume.