Plasmid DNA was purified from 2 to 6 ml of E. coli DH5α or S. aureus RN4220 overnight cultures. For preparation from S. aureus cultures, cells were pelleted, re-suspended in 100 μl TSM buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.5 M sucrose) then treated with 5 μl lysostaphin (2 mg ml−1) at 37 °C for 1 h before treatment with plasmid miniprep reagents from Qiagen. Purification used Qiagen or EconoSpin columns.
Lysostaphin
Manufactured by Qiagen
Lysostaphin is a bacteriolytic enzyme that specifically hydrolyzes the pentaglycine cross-links in the peptidoglycan layer of Staphylococcus aureus cell walls. It is used in various microbiology and molecular biology applications for the lysis of Staphylococcus species.
Automatically generated - may contain errors
2 protocols using lysostaphin
1
Plasmid Purification from E. coli and S. aureus
2
Bacterial Genome Sequencing on Illumina
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Isolates were grown on trypticase soy agar media and DNA was prepared using DNeasy Blood & Tissue Kit as described by the manufacturer (Qiagen, Valencia, CA) with the addition of lysostaphin to the gram-positive extraction protocol from Qiagen. The DNA samples were prepared for multiplexed, paired-end sequencing with a 500 base pair insert using Library Preparation Kit with Standard PCR Library Amplification (KAPA Biosystems, Woburn, MA). A 100 bp read paired-end run was used for all twenty-four isolates on the GAIIx sequencing platform (Illumina, Inc. San Diego, CA).
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