Magnapure lc je379

At the time in which the highest CI value was achieved for each different sample type, the biofilm adhering to the bottom surface of the well was removed as follows: After taking the plate out from the incubator, the supernatant was extracted from the well. Next, two gentle washes with 150 ul of phosphate buffer were performed to remove non-adhered cells. Subsequently, two further exhaustive washes focusing on the bottom surface were carried out until complete dispersion of all adhered material was achieved. Disaggregated biofilm was recovered and stored at −20ºC until processed.
DNA was extracted with the MagnaPure LC JE379 instrument and the MagnaPure LC DNA Isolation Kit (Roche) following the manufacturer´s instructions with some modification to include a strong enzymatic lysis. Specifically, the additional enzymatic lysis step included lysozyme (20 mg/ml, 37°C, 60 min), lysostaphin (2,000 units/mg protein, 37°C, 60 min) and mutanolysin (4,000 units/mg protein, 37°C, 60 min), following reference [24]. The obtained DNAs were eluted in 100 µl of nucleic acid-free water and quantified by fluorometry with the Qubit Kit (Invitrogen).

An aliquot of 250 μl of each saliva sample was thawed and centrifuged at 13.000 rpm for 10 min, and the pellet was resuspended in 100 μl of PBS. Dental plaque samples were mixed by vigorous shaking. Then both kind of samples were immersed in an ultrasounds bath (Selecta) during 45 seg. Before starting DNA extraction with the MagnaPure LC JE379 instrument and the MagnaPure LC DNA Isolation Kit (Roche) following the manufacturer's instructions, an additional enzymatic lysis step was performed as published before (Dzidic et al., 2018 (link)). DNA extracted was quantified in duplicate with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen).