Tianamp bacteria dna extraction kit

Southern blotting was performed to identify intron insertion in the genomic DNA of C. cellulolyticum mutants as described previously [56 (link)]. All positive colonies confirmed by PCR and sequencing were inoculated and cultivated in fresh liquid GS-2 medium until late-log phase. Then 4 mL cultures were centrifuged to obtain cell pellets for C. cellulolyticum genomic DNA isolation using a TIANamp Bacteria DNA Extraction Kit (Tiangen Biotech, Beijing, China). Genomic DNA was digested with EcoRI and BamHI at 37°C overnight, purified using a traditional phenol/chloroform extraction and ethanol precipitation method, separated by 0.8% agarose gel electrophoresis, and finally transferred to a Nylon membrane (Hybond-NX, GE Healthcare, Pewaukee, WI, USA). The intron probe was amplified using primer set Probe-F/R (Additional file 5) and labeled with digoxigenin-11-dUTP. Hybridization and immunological detection were performed according to the manufacturer’s instructions (DIG-High Prime DNA Labeling and Detection Starter Kit I; Roche, Basel, Switzerland).

Molecular identification was performed according to Screening Criteria of Lactic Acid Bacteria for Feeding Aquatic Animals (TCSWSL016-2019). Four isolates were incubated overnight at 37 °C in MRS broth, and then genomic DNA was extracted using TIANamp bacteria DNA extraction kit (TIANGEN, Beijing, China). Molecular identification was performed by amplifying the 16S rRNA gene using universal primers (27F 5′-AGAGTTTGATCCTGGCTCAG -3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′) and previously described polymerase chain reaction (PCR) reaction conditions (Vaz-Moreira et al., 2009 (link)). PCR products were separated by 1.2% agarose gel electrophoresis and confirmed by sequencing (Genewiz, Suzhou, China). The obtained 16S rRNA sequences of the strains were compared with the EzBioCloud databases to identify the species (https://www.ezbiocloud.net/). Phylogenetic tree was constructed based on 16S rRNA sequences by MEGA software (version 7.0) with a Kimura two-parameter model for distance options and a Neighbor Joining (NJ) method for clustering with 1000 bootstrap replicates (Kumar et al., 2016 (link)).

Before DNA extraction, specimens were boiled for 15 min. Brucella DNA was extracted using TIANamp Bacteria DNA extraction kit (TIANGEN Biotech Corporation, Beijing, China) according to the manufacturer’s instructions [5 (link)].

Y. ruckeri genomic DNA was extracted using a TIANamp Bacteria DNA extraction Kit (Tiangen, Beijing, China). The primers OmpF-F1/ OmpF-R1 of the target gene were designed using the Primer Premier 5.0 software based on the Y. ruckeri strain Nr34/85 OmpF gene sequence deposited in GenBank database (HM142671.1, corresponding OmpF protein accession no.: ADK27779.1). OmpF gene was amplified by PCR under the following conditions: 1 cycle of 94°C for 5 min, 30 cycles of 94°C for 1 min, 56°C for 30 s, and 72°C for 90 s, followed by a final extension of 72°C for 10 min. The product of PCR amplification was expected to be about 1098 bp and purified using the Agarose Gel DNA Extraction Kit (TaKaRa), ligated with the pMD19-T using T4 DNA ligase (TaKaRa) and transformed into E. coli DH5α competent cells. The positive recombinant clones were selected on the Amp/LB plate. The recombinant plasmid was identified by PCR under the aforementioned conditions, digested with restriction enzymes NcoI and SacI, and fractionated on 1% agarose gels. DNA sequencing was conducted by TaKaRa Bio Inc. and the sequence was deposited in the NCBI GenBank to obtain accession number. The correct recombinant cloning plasmids were named as T-OmpF.