Minielute pcr purification kits

DNA was extracted from recovered libraries from three of the infections. An aliquot of each recovered library was added to a lysing matrix B tube (MPBiomedicals, Loughborough, UK) containing 25:24:1 phenol:chloroform:isoamyl alchohol and TE, and mechanically disrupted in a Fastprep™ FP120 (MPBiomedicals) at setting 4 for 30 sec. The preparation was centrifuged at 12,000 × g for 5 min, and the supernatant washed twice with chloroform, and precipitated with × 0.7 volumes isopropanol and × 0.1 volumes 3 M sodium acetate. DNA was resuspended in water and sheared by sonicating 3 times for 30 sec each (1 sec on/1 sec off) with a VibraCell® VC300 and a 6 mm probe. Sheared DNA was repaired by incubating with 100 U μl⁻¹ T4 DNA polymerase (Promega, Southampton, UK), 50 mU μl⁻¹ Klenow (Promega), 100 mU μl⁻¹ polynucleotide kinase (New England Biolabs, Hitchin, UK), 2 mM dNTPs all in × 1 T4 DNA ligase buffer (Promega) for 30 min at 20°C. Repaired DNA was purified using QIAquick™ columns (Qiagen, Crawley, UK), and end labelled with A nucleotides by incubating with 0.25 U μl⁻¹ Klenow, 0.2 mM dATP in × 1 Klenow buffer (Promega) for 30 min at 37°C and purified on MiniElute™ PCR purification kits.

DNA and plasmid isolation were performed using standard methods (Ausubel et al., 2002 ). Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs. Chromosomal DNA from all bacteria was isolated using DNeasy tissue kits (Qiagen); plasmid isolations were performed using QIAprep spin miniprep kits (Qiagen). For both kits, S. aureus cells were lysed by adding 25 μg of lysostaphin (Sigma–Aldrich) to the kit lysis buffer and incubating at 37°C for 30 m prior to following the manufacturer’s protocol. DNA fragments were purified using QIAquick mini-elute PCR purification kits, and PCR was performed using GoTaq Green (Promega). Primers used in this study are indicated in Supplementary Table S3. DNA sequencing was performed by automated sequencing technology through Macrogen Co. (Macrogen USA).