2), and suspended in 150 μL of the same buffer. The suspension was then heated to 50°C, and 150 μL of embedding agarose added from the kit at the same temperature. The suspension was then allowed to solidify in molds. Thereafter, the agarose suspension was incubated at 4°C for 20 min. The
agarose blocks were then incubated overnight at 37°C in 540 μL of lysis buffer I (Bio-Rad) containing 20 μL of lysozyme/lysostaphin solution (lysozyme 25 HDAC inhibitors cancer mg/mL, lysostaphin 2 mg/mL; Bio-Rad) and 20 μL of N-acetylmuramidase solution (N-acetylmuramidase SG 5 mg/mL, Dainippon Pharmaceutical, Osaka, Japan). The agarose blocks were washed once with wash buffer (Bio-Rad) and then incubated overnight at 50°C in 520 μL of proteinase K solution (> 23 U/mL). Then, they were then washed five times with wash buffer (1 hr per wash; Bio-Rad). Before restriction enzyme digestion, the agarose blocks were washed twice (1 hr per wash) with 0.1 × wash buffer, and then balanced for 1 hr in an appropriate restriction enzyme buffer. Restriction enzyme digestion with SmaI (TaKaRa) was performed overnight at 30°C. Restriction enzyme digestion with ApeI (TaKaRa) 5-Fluoracil cost and SacII (TaKaRa)
was performed overnight at 37°C. Electrophoresis was carried out using a CHEF DR III System (Bio-Rad) in 1% PFGE certified agarose (Bio-Rad) with 0.5 × tris/borate/EDTA buffer. The pulse time was 1–12 s, current 6 V/cm, temperature 14°C, and running time 22.5 hr. The agarose gel was stained with ethidium bromide (0.5 μg/mL) and visualized under UV light. The PFGE profiles of the strains were then visually compared. TMC0356 genomic DNA was digested with 11 restriction enzymes (Fig. 1). Banding patterns were obtained by digestion with all restriction enzymes except DraI and RsaI. ApaI, SacII, and SmaI were selected because the bands obtained after digesting the DNA with those enzymes were widely separated (from 24 kb to 290 kb). Ten different macrorestriction Tangeritin patterns were
obtained after digestion of genomic DNA of 15 L. gasseri strains with SmaI and separation by PFGE (Fig. 2). Similar banding patterns were obtained for TMC0356, JCM 1031, and JCM 1131; however, a thick band of 42.9 kb was confirmed for TMC0356 but not for JCM1031 and JCM 1131. No other strain showed a banding pattern similar to that of TMC0356. The genomic DNA profiles of the 15 L. gasseri strains digested with SacII are shown in Figure 3. The banding patterns were similar for TMC0356, JCM1031 and JCM 1131; however, a thick band of 42.9 kb was confirmed for TMC0356 but not for JCM1031, JCM 1131. No other strain showed a banding pattern similar to that of TMC0356. The genomic DNA profiles of the 15 L. gasseri strains digested with Apa I are shown in Figure 4. TMC0356, JCM1031 and JCM 1131 showed identical banding patterns, and hence could not be distinguished. A strain (TMC0356F-100) obtained after subculturing TMC0356 in skim milk 100 times was also analyzed by PFGE.