Screening for the presence of the aprX gene in the test strains was carried out using the set of primers SM2F (5′-AAA-TCG-ATA-GCT-TCA-GCC-AT-3′)/SM3R (5′-TTG-AGG-TTG-ATC-TTC-TGG-TT-3′) with an amplification product of approximately 850 bp, as described by Marchand et al. [20 (link)]. DNA was extracted using the Microlysis kit (Aurogene, Rome, Italy) following the manufacturer’s instructions. The PCR reaction was performed in an Eppendorf Mastercycler nexus (Eppendorf AG, Hamburg, Germany), using the PCR Master Mix (2X) (Thermo Fisher Scientific, Walthman, MA, USA). The cycle parameters were 5 min at 95 °C for initial denaturation followed by 30 cycles with denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C, extension for 1 min at 72 °C, and a final elongation step of 72 °C for 8 min.
Microlysis kit
Manufactured by Aurogene
Sourced in Italy
The Microlysis kit is a laboratory equipment designed for the analysis of small-scale samples. It provides a controlled environment and the necessary tools to perform precise and accurate micro-scale experiments and measurements.
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Lab products found in correlation
2 protocols using microlysis kit
1
Screening for aprX Gene Presence
2
Isolation and Identification of Psychrotrophic Bacteria from Raw Milk
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Fourteen psychrotrophic strains were isolated from the six samples of raw milk. Samples were serially diluted in quarter-strength Ringer's solution (Scharlau Microbiology, Barcelona, Spain), inoculated into Penicillin-Pimaricin (PP) (Biolife, Milan, Italy) agar supplemented with PP Pseudomonas supplement (Biolife) and incubated aerobically at 30 °C for 24-48 h. The colonies with different morphologies were isolated and cultured in Brain Heart Infusion (BHI) broth (Scharlau Microbiology) and purified by streaking repeatedly on PP agar. The 14 isolates were cultivated routinely overnight at 30 °C in BHI broth and preserved in litmus milk at -18 °C.
Genomic DNA was extracted from overnight cultures using the Microlysis kit (Aurogene Rome, Italy) following the manufacturer's instructions. Strain identification was performed by partial 16S rRNA gene and rpoB gene sequencing according to McCabe et al. (1995) and Sajben et al. (2011) (link).
The obtained PCR products were sent to Macrogen Europe (Amsterdam, the Netherlands) for sequencing and sequences were analyzed with NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).
Genomic DNA was extracted from overnight cultures using the Microlysis kit (Aurogene Rome, Italy) following the manufacturer's instructions. Strain identification was performed by partial 16S rRNA gene and rpoB gene sequencing according to McCabe et al. (1995) and Sajben et al. (2011) (link).
The obtained PCR products were sent to Macrogen Europe (Amsterdam, the Netherlands) for sequencing and sequences were analyzed with NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).
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