Tru1I (MseI) (10 U/µL)

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	The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank. Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in previous work [13].

Protocol tips

The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank. Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in previous work [13].

Publication protocol

Nine isolates for which definitive species identification was not obtained using MALDI-TOF MS (L. johnsonii/L. gasseri, L. crispatus/L. ultunensis, or L. oris/L. antri) were identified using Amplified Ribosomal DNA Restriction Analysis of 16S rDNA (16S-ARDRA). Isolation of bacterial genomic DNA from lactobacilli and amplification of 16S rDNA were performed according to the protocol described in our previous work [13].Eight reference Lactobacillus strains were used in the experiment: L. antri LMG 22111, L. crispatus LMG 9479, L. gasseri LMG 13134, L. gasseri ATCC 19992, L. johnsonii LMG 18195, L. johnsonii LMG 9436, L. oris LMG 9848 and L. ultunensis LMG 22117. The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank.Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in a previous work [13].

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