Strains were grown overnight on Trypticase Soy Agar (BD Biosciences, Sparks, MD) at 37°C to obtain isolated colonies. Individual colonies were selected to start an overnight culture of Trypticase Soy Broth (BD Biosciences, Sparks, MD). After 12–18 hours of growth, 750 μl of the liquid culture was pelleted and the supernatant was removed. The pelleted cells were stored at -80°C until DNA extraction. To isolate DNA, each pellet was resuspended in 200 μl 1x Phosphate Buffered Saline with 0.2 M EDTA. To lyse the cells, the following were added to each suspension: 12 μl of Lysozyme solution (Sigma, St. Louis, MO), 1 μl RNase (Roche, Mannheim, Germany), 7.5 μl Lysostaphin solution (Sigma, St. Louis, MO), and 7.5 μl Mutanolysin solution (Sigma, St. Louis, MO). The cells were then incubated for 1 hour at 37°C. Forty microliters of Proteinase K (Roche, Mannheim, Germany) was added and the suspension was incubated overnight at 55°C. The following day, a Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) was used to isolate DNA according to the manufacturer’s protocol. The Elution Buffer containing DNA samples was centrifuged for 5 minutes at 8000xg to remove visible debris. The supernatant was transferred into a clean 1.5 mL tube and stored at 4°C until further analysis.
Mutanolysin solution
Manufactured by Merck Group
Mutanolysin solution is a bacterial enzyme that cleaves the peptidoglycan layer of gram-positive bacterial cell walls. It is commonly used in laboratory settings for the isolation and purification of bacterial DNA and RNA.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Roche (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Trypticase soy agar, by BD (1 mentions)
Trypticase soy broth (tsb), by BD (1 mentions)
Lysozyme solution, by Merck Group (1 mentions)
Lysostaphin solution, by Merck Group (1 mentions)
Mgcl2, by Scharlab (1 mentions)
2 protocols using mutanolysin solution
1
Bacterial DNA Extraction Protocol
2
Mutanolysin Sensitivity Assay for Lactobacillus
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Mutanolysin sensitivity was evaluated on Lb. sakei cells as described by Ouzari et al. [22] (link). Cells were harvested in late exponential phase (OD600 = 0.8–1.0) and suspended to an OD600 of approximately 0.5, using MES (2-N-Morpholino-ethansulfonic acid) (Sigma) buffer (50 mM, pH 6.0) supplemented with 1 mM MgCl2 (Scharlau). A volume of 3 ml aliquot of cell suspension was then equilibrated at 37 °C after which the OD600 was measured. Subsequently, 5 μl of mutanolysin solution (Sigma) (150 U ml−1) prepared in TES buffer (N-Tris-hydroxymethyl-2-aminoethansulfonic acid) (Sigma) (50 mM, pH 7.0), 1 mM MgCl2 (Scharlau), was added to the cell suspension. The mixture was incubated at 37 °C for 20 min and the OD600 was then measured. The mutanolysin activity was expressed as the percentage decrease of the OD600.
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