For 16 S rRNA sequencing analysis, genomic DNA was extracted from the isolated strain with the mini BEST Bacterial Genomic DNA Extraction Kit (TAKARA BIOTECHNOLOGY, LTD.) according to the manufacturer’s instructions. The 16 S rRNA gene sequence was amplified using the universal bacterial primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) and cloned in the pMD18-T vector (TAKARA BIOTECHNOLOGY, LTD.) for sequencing. The obtained sequence was analysed against the NCBI and EzTaxon databases to identify the most closely related species. Then, a phylogenetic tree was constructed with MEGA (version 4.0)28 (link).
Minibest bacterial genomic dna extraction kit
Manufactured by Takara Bio
Sourced in China, Japan
The MiniBEST Bacterial Genomic DNA Extraction Kit is a laboratory product designed to efficiently extract high-quality genomic DNA from bacterial samples. It provides a reliable and streamlined process for isolating DNA, which is a crucial step in various molecular biology and genomics applications.
Automatically generated - may contain errors
Lab products found in correlation
Pmd18 t vector, by Takara Bio (1 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (1 mentions)
Anti gapdh antibody, by Bioworld Technology (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Tcdca, by Merck Group (1 mentions)
Milli q ultrapure water purification system, by Merck Group (1 mentions)
Gcdca, by Merck Group (1 mentions)
Tβmca, by Merck Group (1 mentions)
G lca, by Steraloids (1 mentions)
G udca, by Steraloids (1 mentions)
G dca, by Steraloids (1 mentions)
T β mca, by Steraloids (1 mentions)
T udca, by Steraloids (1 mentions)
T hdca, by Steraloids (1 mentions)
T lca, by Steraloids (1 mentions)
Dss mw 36 000 50 000, by MP Biomedicals (1 mentions)
Mouse total bile acids kit, by Crystal Chem (1 mentions)
Sybr green pcr master mix, by Qiagen (1 mentions)
Dulbecco s phosphate buffered saline dpbs 1x, by Thermo Fisher Scientific (1 mentions)
Baclight red bacterial stain, by Thermo Fisher Scientific (1 mentions)
8 protocols using minibest bacterial genomic dna extraction kit
1
Identification of Bacterial Species via 16S rRNA Sequencing
2
Comprehensive Bile Acid Profiling Protocol
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HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Nepean, Ont., Canada). Ultrapure water was prepared by the Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). AOM was obtained from Sigma-Aldrich (St. Louis, MO, USA), and DSS (MW 36000-50000) was purchased from MP Biomedicals (Santa Ana, CA). Ammonium acetate, formic acid, glacial acetic acid and the other reagents (analytical grade) were purchased from Nanjing Chemical Factory (Nanjing, China). ‘Mouse Total Bile Acids Kit’ was purchased from Crystal Chem Inc (Downers Grove, United States). ‘MiniBEST Bacterial Genomic DNA Extraction Kit’ was from TaKaRa Bio Inc (Dalian, China). Anti-FXR, FGF15 and CYP7A1 antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH antibody was from Bioworld Technology (St. Louis Park, MN, USA).
CA, CDCA, DCA, and LCA, as well as their glycine [38 (link)] and taurine [45 (link)] conjugates, G-CA, G-CDCA, T-β-MCA, T-CA, T-CDCA, T-DCA, and internal standard dehydrocholic acid (dhCA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). α-MCA, UDCA, HDCA, G-LCA, G-UDCA, G-DCA, T-β-MCA, T-UDCA, T-HDCA and T-LCA were purchased from Steraloids, Inc. (Newport, Rhode Island, USA).
CA, CDCA, DCA, and LCA, as well as their glycine [38 (link)] and taurine [45 (link)] conjugates, G-CA, G-CDCA, T-β-MCA, T-CA, T-CDCA, T-DCA, and internal standard dehydrocholic acid (dhCA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). α-MCA, UDCA, HDCA, G-LCA, G-UDCA, G-DCA, T-β-MCA, T-UDCA, T-HDCA and T-LCA were purchased from Steraloids, Inc. (Newport, Rhode Island, USA).
3
Bacterial DNA Extraction and Quantification
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We purchased HisPur™ Ni-NTA Magnetic Beads and BacLight Red Bacterial Stain from THERMO FISHER SCIENTIFIC (USA). SYBR Green PCR master mix was purchased from QIAGEN (Germany). Dulbecco’s phosphate buffered saline (DPBS, 1X) was purchased from GIBCO (Life Technologies). Human plasma (pooled normal, K2 EDTA) was purchased from INNOVATIVE RESEARCH INC. (USA). A Mini-BEST bacterial genomic DNA extraction kit was purchased from TAKARA (Japan). Primer sets for bacterial DNA amplification by real-time PCR were purchased from BIONEER INC. (Korea), and their sequences are summarized in Table S1 .
4
Bacterial DNA Extraction Methods
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Two DNA extraction methods, NaOH-based and Kit-based, were employed to extract bacterial genomic DNA. The NaOH-based method was used to crudely extract the genomic DNA of V. vulnificus. Briefly, 50 μL of V. vulnificus suspension was added to 200 μL of 0.5 M NaOH solution and incubated at room temperature for 3 min. After being diluted 20-fold with nuclease-free water (Qiagen, Germany), 2 μL of cell lysate was used as template for the RAA assay. A MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, China) was also used to extract bacterial genomic DNA according to the user manual.
5
Bacterial 16S rRNA Gene Sequencing
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Isolated strains were grown in 10 mL Luria-Bertani liquid medium with 180 rpm agitation at 30 °C, for 24 h. The bacterial DNA was extracted using the MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, Japan). We used the universal primers of the 16S rRNA gene to amplify the 16S rRNA gene fragments. The resulting fragments were sequenced by Shanghai Majorbio Bio-pharm Technology Co. Ltd (China), and compared in the NCBI (http://www.ncbi.nlm.nih.gov/ ) and EzTaxon (http://www.ezbiocloud.net/ ) databases.
6
Bacterial DNA Isolation from Dental Unit Water
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The 250 mL water samples from air/water syringes of 60 DCUs before daily dental practice were filtered through a 0.2-μm pore polycarbonate filter (Millipore, Billerica, MA, USA), which was then stored in phosphate-buffered saline at 4 °C. Microorganism precipitates were obtained by centrifugation at 12,000 rpm for 10 min; the filter membrane was discarded. Bacterial genomic DNA was extracted using a Takara MiniBEST Bacterial Genomic DNA Extraction Kit (Takara, Dalian, China), following the manufacturer’s protocol. High-quality DNA with OD260/280 = 1.8–2.0, concentration >5 ng/μL, and no degradation with agarose gel electrophoresis (from 17 air/water syringe water samples) was stored at −80 °C until being used for molecular applications.
7
Genomic Analysis of P. gingivalis Fimbriae
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Periodontal examination was performed carefully. The number of teeth was recorded. Subgingival plaque samples were then processed from the deepest periodontal sites with periodontal depth ≥5 mm with sterile Gracycurettes. Bacterial genomic DNA was isolated from these samples with MiniBEST Bacterial Genomic DNA Extraction Kit (Takara). The isolated DNA was stored at −20°C. P.gingivalis fimA genotype was determined by PCR method. To analyze fimA gene, the specific primers for each subtype described by Hayashi et al [24] (link) were used. All PCR products were viewed by electrophoresis. In each sample processing, we set controls to avoid false positives and negatives.
8
Assessing Genomic DNA and RNA Integrity
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As with the method for measuring the zeta potential, the same number of cell pellets were exposed to different concentrations of aqueous ozone. The samples were then centrifuged and their genomic DNA was extracted using a MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, China), and the total RNA was extracted by TRIzol reagent (Invitrogen, USA). The nucleotide concentrations were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The integrity of the nuclei acid was verified using 1% (w/v) agarose gel electrophoresis by loading the same volume of gDNA or total RNA.
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