Genomic DNA Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

PCR amplification, sequencing of spa gene and Spa typing
DNA was extracted from all S. aureus strains, which were grown overnight in nutrient agar (Merck/Germany) at 37 °C using the Fermentas DNA kit, according to the manufacturer's instruction (Fermentas, USA). The primers used for amplification of the X region of the spa gene in this study were as follows: spa-1113f (5-TAAAGACGATCCTTCGGTGAGC-3) and spa-1514r (5-CAGCAGTAGTGCCGTTTGCTT-3) [9]. For the characterization of nucleotide sequences based on the strands, the PCR products were run on 1.5% agarose gels, then sent for sequencing (Bioneer, Korea). The spa typing and evaluation of spa types from these S. aureus strains were performed using the spa database website (http://www.ridom.de/spaserver). The BURP algorithm was used to determine spa clonal complexes (CCs) using the StaphType software v. 1.5 (Ridom GmbH, Würzburg, Germany). When the relationship of various spa types was calculated, insertions, deletions and any recombination in repeats were accounted for. Two default parameters are offered by BURP for clustering analysis: (1) spa types that are shorter than “5 repeats” were excluded, and (2) spa types with a maximum number of costs less than or equal to “4 repeats” were clustered into the same group. A cluster consisting of two or more related spa types was regarded as a clonal complex. A singleton was defined as a spa type that was not grouped into a clonal complex.



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Manufacturer protocol

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