Grouping of the RAPD-PCR profiles was carried out using the BioNumeric 5.1 software package (Applied Maths, Sint-Martens-Latem, Belgium). The resulting dendrogram was created by the unweighted pair group method with arithmetic averages (UPGMA) cluster analysis; strains sharing the same number and the same size of PCR bands were considered genetically identical, while any relationship >90% and <100%, was defined as closely related.
Bionumeric 5
Manufactured by bioMérieux
Sourced in Belgium
BioNumeric 5.0 is an automated microbiology platform designed for colony counting and identification. It utilizes advanced image analysis technology to efficiently enumerate and classify bacterial colonies on culture media.
Automatically generated - may contain errors
Lab products found in correlation
7 protocols using bionumeric 5
1
Genetic Fingerprinting of Microbial Strains
2
Genetic Diversity Analysis of Prototheca Strains
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The RAPD-PCR was used to explore the genetic diversity of the 49 Prototheca strains. The DNA of P. stagnora ATCC 16528 (STAG) and P. ulmea ATCC 50112 (ULM) were included in the RAPD-PCR analysis. The RAPD-PCR assay was developed by screening 10 primers: M13 (5 -GAGGGTGGCGGTTCT-3 ); OPAA10 (5 -GGACTACCAGGGTATCTAAT-3 ; as described by Decimo et al., 2014) ; D11344 (5 -AGTGAATTC-GCGGTCAGATGCCA-3 ), D8635 (5 -GAGCGGC-CAAAGGGAGCAGAC-3 ; according to Morandi et al., 2013) ; 208 (5 -ACGGCCGACC-3 ), 272 (5 -AGC-GGGCCAA-3 ; as reported by Saitou et al., 2010) ; HLWL85 (5 -ACAACTGCTC-3 ; according to Wulff et al., 2006) ; OPA-4 (5 -AATCGGGCTG-3 ); OPA-13 (5 -CAGCACCCAC-3 ); and OPA-18 (5 -GAGAGC-CAAC-3 ; Gómez and González, 2001) . The amplification with OPA-4, OPA-13, and OPA-18 started with an initial denaturation at 94°C for 2 min, followed by 55 cycles at 94°C for 1 min, 40°C for 2 min, 72°C for 2 min, and final extension at 72°C for 5 min. Grouping of the RAPD-PCR profiles was obtained with the BioNumeric 5.1 software package (Applied Maths, Kortrjik, Belgium) using the unweighted pair group method using arithmetic averages (UPGMA) cluster analysis.
The reproducibility value of the RAPD-PCR assay, calculated from 2 repetitions of independent amplification of Prototheca type strains, was higher than 90%.
The reproducibility value of the RAPD-PCR assay, calculated from 2 repetitions of independent amplification of Prototheca type strains, was higher than 90%.
3
Rapid Bacterial Strain Identification
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LAB strains were typed by random amplification of polymorphic DNA-PCR (RAPD) typing with primers M13 (5′-GAGGGTGGCGGTTCT-3′) and LP1 (5′ACGCGCCCT-3′). An annealing temperature of 38 and 48 °C for M13, and LP1 respectively, and an amplification protocol of 35 cycles were used. The PCR products were analyzed by electrophoresis and photographed as reported earlier. Grouping of the RAPD-PCR profiles was obtained with the BioNumeric 5.0 software package (Applied Maths, Kortrijk, Belgium), following the unweighted pair-group method with arithmetic averages cluster analysis. The value for the reproducibility of the assay, evaluated by the analysis of repeated DNA extracts of representative strains was > 93%.
4
Molecular Profiling of S. aureus Virulence
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The DNA was amplified to investigate the presence of 26 factors that can contribute in different ways to S. aureus pathogenicity and therefore influence the management of the disease. In this study genes encoding enterotoxins (from sea to sel), leucocidins (lukE, lukSF-PV, lukE-lukD, lukM), the acquisition of methicillin resistance (mecA) and genes related to host invasion (clfA, fmtB, cna, eta, etb) or to factors that have the potential to interfere with host defense mechanisms (tsst, scn, chp, sak) were analyzed using primers and protocols described in literature and listed in Table 11 . The amplified PCR fragments were visualized on 2% agarose gel electrophoresis (GellyPhor, Euroclone, Milan, Italy), stained with ethidium bromide (0.05 mg/mL; Sigma Aldrich, Milan, Italy), and visualized by UV transilluminator (BioView Ltd., Nes Ziona, Israel). A 100 bp DNA ladder (Finnzymes, Espoo, Finland) was included in each gel.
Grouping of the RS-PCR profiles and the virulence factors was obtained with the BioNumeric 5.0 software package (Applied Maths, Kortrjik, Belgium) using the UPGMA (unweighted pair group method using arithmetic averages) cluster analysis.
Grouping of the RS-PCR profiles and the virulence factors was obtained with the BioNumeric 5.0 software package (Applied Maths, Kortrjik, Belgium) using the UPGMA (unweighted pair group method using arithmetic averages) cluster analysis.
5
Genomic DNA Extraction and RAPD-PCR Profiling of Enterococcus lactis
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Genomic DNA used for RAPD-PCR amplification was extracted from overnight culture of E. lactis 4CP3 in M17 broth at 30°C according to Cremonesi et al., 2006 [25 (link)]. RAPD-PCR amplification was realised using the universal primers M13 and D8635 as described by Andrighetto et al., 2001 [26 (link)]. Amplification products were separated by electrophoresis on agarose gel (1.5%) in 1 × TAE buffer at 100 mV for 99 min. The gels were stained in ethidium bromide and photographed on a UV transilluminator. Photo-positives were scanned into a computer and were analysed using the BioNumeric 5.0 software package (Applied Maths NV, Sint-Martens-Latem, Belgium). Grouping of the RAPD-PCR patterns was performed using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) cluster analysis. The reproducibility value of the RAPD-PCR assay, calculated from two repetitions of independent amplification of type strains, was higher than 90%. The RAPD-PCR profiles obtained with both primers (M13 and D8635) were analysed together to obtain a single dendrogram.
6
Rapid Microbial Community Profiling of Whey
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Randomly amplified polymorphic DNA-PCR analysis was performed to evaluate the heterogeneity of the microbial community of NWC as previously reported by Zago et al. (2021) (link). The DNA was directly extracted from 1 mL of NWC according to Cremonesi et al. (2006) (link), and RAPD-PCR was applied to the total DNA of each whey sample using 4 primers: M13 (Huey and Hall, 1989) (link) and OPA-4, OPA-13, and OPA-18 (Morandi et al., 2016) (link). The RAPD-PCR fingerprints were imported into BioNumeric 5.0 (Applied Maths) and analyzed using composite data set and UPGMA cluster analysis.
7
Evaluating Cholesterol-Lowering Strains via RAPD PCR
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To evaluate the implantation capacity of the cholesterol-lowering strains, the random amplified polymorphic DNA (RAPD) PCR analysis was used to characterize the isolates from the experimental cheeses. The RAPD PCR reactions were performed with primers M13 (5′-GAGGGTGGCGGTTCT-3′; McCabe et al., 1995) . Grouping of the RAPD PCR profiles was obtained with the BioNumeric 5.0 software package (Applied Maths, Kortrjik, Belgium) using unweighted pair group method using arithmetic averages (UPGMA) cluster analysis. The reproducibility value of the RAPD PCR assay, calculated from 2 repetitions of independent amplification of the LAB type strains, was higher than 90%. Thus, isolates showing a similarity coefficient equal to or higher than 95% to the inoculated strain were considered as being identical.
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