RAPD-PCR reactions were performed using the OLP 13 primer (5’-ACCGCCTGCT-3’) (Zare et al., 2019 (link)), and a 25μl reaction mix, containing 3mM MgCl2 (Bio Line, London, UK), 0.5 mM dNTPs (New England BioLabs, UK), 1 µM of primer, 1 U of Taq DNA polymerase (Bio Taq DNA polymerase, Meridian Bioscience, USA) in 1X PCR buffer (Meridian Bioscience, USA) and 3 μl of template dsDNA, was prepared. PCR was performed as follows: 1 cycle at 94°C for 5 min, followed by 40 cycles at 93°C for 1 min, 37°C for 1.5 min, and 72°C for 1 min, with a final extension at 72°C for 7 min (Mastercycler pro, Eppendorf, Germany). Amplification products were solved by electrophoresis on 1% agarose-TBE gel, stained with 5µl of “Midori Green Advance DNA Stain” (Nippon Genetics Europe GmbH, Germany), visualized under UV transilluminator (UVP Inc., Cambridge) and photographed with DigiDoc-It (UVP, Cambridge) system. To guarantee the reproducibility of RAPD-PCR profiles, the reactions were carried out in triplicate.
Digidoc it
Manufactured by Analytik Jena
Sourced in United States
The DigiDoc-It is a compact and versatile documentation system for gel analysis. It features a high-resolution camera and integrated UV transilluminator for capturing images of electrophoresis gels, blots, and other samples. The system provides a simple and user-friendly interface for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Agarose gel, by Thermo Fisher Scientific (2 mentions)
Biotaq dna polymerase, by Meridian Bioscience (1 mentions)
Mgcl2, by Meridian Bioscience (1 mentions)
Mastercycler pro, by Eppendorf (1 mentions)
Midori green advance dna stain, by Nippon Genetics (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Molecular size markers, by Thermo Fisher Scientific (1 mentions)
Abi prism 3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Pcr clean up system, by Promega (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Big dye terminator cycle sequencing ready reaction version 3, by Thermo Fisher Scientific (1 mentions)
Wizard sv gel, by Promega (1 mentions)
Mastercycler personal, by Eppendorf (1 mentions)
5 protocols using digidoc it
1
RAPD-PCR Amplification Protocol for DNA Profiling
2
Species-Specific PCR for Identification
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Species-specific PCR was performed as described elsewhere (Hogardt et al., 2007 (link)). Negative and positive control PCRs were employed for every experiment. PCR products were visualized by electrophoresis in a 2% agarose gel (Invitrogen Corporation, CA), stained with ethidium bromide (EtBr; Invitrogen Corporation), and captured with a DigiDoc-It (UVP, Cambridge, United Kingdom) photographic system. Bands of 163 bp were considered positive for A. xylosoxidans identification.
3
RAPD Amplification and Gel Electrophoresis
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The RAPD amplification mixture and cycling conditions were described elsewhere (Lambiase et al., 2006 (link)). The primer used was the 270 (5′-TGCGCGCGGG-3′). RAPD products were separated by electrophoresis in 1.5% agarose gel (Invitrogen Corporation, CA). Molecular size markers (Invitrogen Corporation) and negative control were included in all gels. Gels were stained with EtBr (at 0.5 μM) (Invitrogen Corporation) and captured with a DigiDoc-It (UVP) photographic system.
4
Pfcrt Gene Amplification and Sequencing
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The DNA from 1 mL blood samples was extracted using a QIAamp™ DNA Blood Midi Kit (QIAGEN), according to the manufacturer’s instructions. PCRs were performed to amplify the pfcrt fragment gene according to previously described protocols [25 (link)]. PCR products were analyzed by electrophoresis on 2% agarose gel and visualized under a UV transilluminator (DigiDoc-It, UVP, Upland, CA, USA). Each PCR product was purified using Wizard™ SV Gel and the PCR Clean-Up System (Promega, WI, USA), following the manufacturer’s procedure. Purified DNA sequencing was carried out through Big Dye™ Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems, Carlsbad, CA, USA), with 3.2 μM of forward and reverse PCR primers. DNA sequences to investigate C72S , M74I , N75E/D , and K76T were determined using an ABI Prism DNA Analyzer™ 3730 (Applied Biosystems, CA, USA), on the Fiocruz Genomic Platform PDTIS/Fiocruz RPT01A. Nucleotide sequences were aligned using the ClustalW multiple sequence aligner in the BioEdit software [26 ]. The PF3D7_1343700 strain was used as a reference sequence (from PlasmoDB: http://www.plasmoDB.org , accessed on 20 March 2023). DNA sequences were deposited in GenBank (the NIH’s genetic sequence database; www.ncbi/nlm/nih.gov/GenBank , accessed on 21 March 2023) with the accession numbers OQ672386-OQ672451.
5
Virulence Gene Detection in Bacillus
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All strains were evaluated for the presence of sequences associated with virulence genes by PCR. Genes encoding for cereulide (ces) (Ehling-Schulz et al., 2005) , cytotoxin K (cytK) (Ehling-Schulz et al., 2006) , sphingomyelinase (sph) (Hsieh et al., 1999) ; the components of hemolysin BL (hblA, hblB, hblC, hblD) (Granum et al., 1996; Ryan et al., 1997 ) and the non-hemolytic complex (nheAB) (Guinebretière et al., 2010) were studied. Primers used and conditions of amplification are listed in Table 1. DNA amplifications were performed in a thermal cycler (Mastercycler personal; Eppendorf Hamburg, Germany). The amplification products were separated in 1.6% (W/V) agarose gel in 0.5× TBE buffer, stained with ethidium bromide, and visualized with a UV transilluminator (UVP, Upland, California, USA). Gel images were digitalized by using a digital image capture gel documentation system (DigiDoc-It, UVP, v.1.1.25, Upland, California, US).
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