Fastprep instrument fp 24

0.2 g of each smokeless tobacco product was transferred under sterile conditions to a Lysing Matrix B tube (MP Biomedicals, Solon, OH), and enzymatic lysis was initiated by adding to each tube: 1 ml ice cold 1X PBS buffer (molecular grade; Gibco, Life Technologies, NY), 5 μl of 10 mg/ml lysozyme (Sigma-Aldrich, MO), 5 μl of 5 mg/ml lysostaphin (Sigma-Aldrich, MO) and 15 μl of 1 mg/ml mutanolysin (Sigma-Aldrich, MO). Tubes were then incubated at 37°C for 30 min, after which 10 μl of 20 mg/ml Proteinase K (Invitrogen by Life Technologies, NY) and 50 μl of 10% w/v SDS (BioRad) were added to each tube, followed by incubation at 55°C for 45 min. Samples were then mechanically lysed using a FastPrep Instrument FP-24 (MP Biomedicals, CA) at 6.0 m/s for 40 secs. The resulting lysate was then briefly centrifuged and DNA was purified using the QIAmp DSP DNA mini kit 50, v2 (Qiagen, CA), according to the manufacturer’s protocol. In order to remove any potential PCR inhibitors, the extracted DNA was further purified by precipitation using 3M sodium acetate (1/10 volume) and 100% ethanol (2.5 volumes), followed by overnight incubation at −20°C. The DNA was then pelleted, washed with 80% ethanol and resuspended in Tris-EDTA buffer.