Easy dna purification kit

Gene transfer between P. gingivalis cells and vesicles was examined using vesicles isolated from a mutant strain generated from P. gingivalis 49417 by introducing a 2.1-kb ermF-ermAM cassette into the fimA gene. Since the erm resistant gene was detectable in these vesicles, we used these vesicles as donors in a DNA transfer assay. In brief, P. gingivalis 33277 was anaerobically cultured in TSB at 37° for 6 h to reach an optical density at 600 nm (OD₆₀₀) of 0.5. Cells (10⁸) were pelleted, resuspended, and mixed with the vesicles (2.5 μg) in 1 ml of fresh TSB. The suspension was incubated for 24 h, and recipients were selected by plating the bacterial suspension on TSB blood plates supplemented with erythromycin (5 μg/mL). After culturing for 5–7 days, the colonies were counted and collected for future PCR analysis. To confirm that the erm resistant gene was indeed incorporated into the fimA gene, P. gingivalis chromosomal DNA was isolated from the recipients using an Easy-DNA Purification Kit (Invitrogen). PCR was performed using DNA isolated from the recipients as a template, while the primers corresponded to sequences of the fimA gene.

Tissue samples were stored in 70 % ethanol until DNA extraction. After washing with Dulbecco’s phosphate-buffered saline (PBS) (Sigma, Cat. #D8567), gDNA was extracted using a lysis buffer (500 μg/mL Proteinase K [Merck, Cat. #1.24568.0500], 40 mM dithiothreitol [Wako, Cat. #048-29224], 0.08 % SDS and 1 × PCR buffer [Takara, Cat. #R001A]) at 50 °C for 16 h and then extracted with phenol-chloroform, and precipitated with ethanol using the standard procedures [39 ]. Semen samples were washed with 0.11 M sodium citrate and PBS twice before gDNA extraction. gDNA of whole blood was extracted using the Easy-DNA Purification Kit (Invitrogen, Cat. #K1800-01). DNA quality was confirmed by agarose electrophoresis.