Hiyield gel pcr fragments extraction kit

Lineages representative of the clusters obtained by PFGE were selected for MLST. Five MRSA isolates obtained PFGE-SmaI and four MSSA isolates obtained by PFGE-ApaI were chosen.
MLST was performed according to the protocol of Enright et al. [36 (link)] by amplification and sequencing of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL). The PCR products were purified using the HiYield™ Gel/PCR Fragments Extraction Kit and the sequencing reactions were performed in an ABI3500 8-capillary sequencer (50 cm) using POP7 as polymer (Applied Biosystems). The BioNumerics 7.6 program (Applied Maths, Belgium) was used for visualization of the sequences (electropherogram). The sequences were analyzed and compared to an online database (http://www.mlst.net) (2004).

MLST was performed according to standards described in 2000 by Enright et al. [35 (link)]. Each primer pair amplifies an internal fragment of the housekeeping gene (about 500 bp) (Table 5): carbamate kinase dehydrogenase (arcC), chiquimate dehydrogenase (aroE), glycerol kinase (glpF), guanylatoquinase (gmk_), phosphate acetyltransferase (pta_), triosephosphate isomerase (tpi_), and acetyl coenzyme A acetyltransferase (yqiL).
Purification was performed by the HiYield ™ Gel/PCR Fragments Extraction Kit and reactions were performed on an ABI3500 8 capillary (50 cm) sequencer using POP7 (Applied Biosystems) as the polymer. Sequence visualization (electrophoresis) was performed by Mega, Laser Gene and Bionumerics (version 7.1, Applied Maths, Belgium). The analysis and comparison of the sequences were performed on an internet database (http://www.mlst.net).