Bacterial DNA Extraction from Uterine and Vaginal Samples

Bacterial DNA was extracted as described previously (21 (link)). After thawing, 2 ml of uterine and vaginal samples were mixed with 20 ml of sterile saline solution and centrifuged at 8,000 g for 15 min. Pellets were then suspended in 800 μl 10 mM Tris-HCl and 10 mM EDTA buffer containing lysozyme (Sigma-Aldrich Co., LCC, Missouri, USA) (Final concentration: 15 mg/ml). Mixtures were incubated at 37°C for 1 h. Purified achromopeptidase (Wako Pure Chemical Inc., Osaka, Japan) (Final concentration: 2,000 U/ml) was added and incubated 37°C for 30 min. Finally, proteinase K (Takara-Bio Inc., Shiga, Japan) (Final concentration: 1 mg/ml) with 20% sodium dodecyl sulfate (Sigma-Aldrich Co., LCC) was added and incubated at 55°C for 1 h. The DNA was isolated with phenol:chloroform:isoamyl alcohol (25:24:1, v/v), washed twice with 75% ethanol, and dissolved in 100 μl TE buffer. RNase A (Nippon Gene Co., Ltd., Tokyo, Japan) (Final concentration: 0.1 mg/ml) was added to the mixture, and incubated at 37°C for 1 h. Subsequently, the DNA was purified using a High Pure PCR Template Kit (Roche Inc., Basel, Switzerland) according to the manufacturer's instructions. Elution was performed in 50 μl of TE buffer and then samples were stored at −20°C until further analysis.