Genotypic identification was carried out by partial 16S rRNA gene sequencing. Genomic DNA was extracted according to Pospiech and Neumann.30 (link) Oligonucleotide primers (PLB16, 5-AGAGTTTGATCCTGGCTCAG-3, and MLB16, 5-GGCTGCTGGCACGTAGTTAG-3) were used to amplify the variable (V1) region of the 16S ribosomal RNA gene according to the protocol described by several authors.31 (link), 32 (link), 33 (link) PCR products were electrophoresed in 1% (wt/vol) agarose gels, stained and visualized as described above. Amplicons were excised from the gel and purified using a GFX PCR DNA gel band purification kit (GE Healthcare, UK). Purified PCR products were sequenced at CERELA-CONICET, Tucumán, Argentina, by using an ABI 3130 DNA sequencer (Applied Biosystems, Foster, CA). rRNA gene sequence alignments were performed using the multiple sequence alignment method34 , 35 (link) and identification queries were fulfilled by a BLAST search36 (link) in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/ ) and in the Ribosomal Database Project.37 (link)
Gfx pcr dna gel band purification kit
Manufactured by GE Healthcare
Sourced in United Kingdom
The GFX PCR DNA gel band purification kit is a lab equipment product designed to extract and purify DNA fragments from agarose gels. It is used to isolate and concentrate specific DNA bands after gel electrophoresis for downstream applications.
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Lab products found in correlation
Abi 3130 dna sequencer, by Thermo Fisher Scientific (2 mentions)
3100 avant genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Abi prism bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100, by Thermo Fisher Scientific (1 mentions)
Puregene dna purification kit, by Qiagen (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using gfx pcr dna gel band purification kit
1
Partial 16S rRNA Gene Sequencing for Bacterial Identification
2
DNA Sequencing Purification Protocol
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Amplification products were purified by electrophoresis (Sambrook, Fritsch & Maniatis, 1989 ), one-band DNA fragments were extracted from the gel and purified using the GFX™ PCR DNA, Gel Band Purification Kit (GE HealthCare, Little Chalfont, UK), or Evrogene Cleanup Mini (Russia), and then used as a template in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) following the standard protocol provided for 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fragment sequences were determined at the Genom Center (Engelhardt Institute of Molecular Biology RAS, Moscow, Russia).
3
16S rRNA Gene Sequencing and Phylogenetic Analysis
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An isolated colony was used for genomic DNA extraction, following the method described by Pospiech et al.[24 (link)], with some modifications. The amplification of the 16S rRNA gene was performed through polymerase chain reaction (PCR). The products were analyzed and visualized by agarose gel electrophoresis and purified with mini columns (GFX PCR DNA & gel band purification kit, GE Healthcare) in an ABI3500XL Series automatic sequencer (Applied Biosystems) according to the manufacturer’s specifications. The detailed protocol conditions used in this section can be found in Flores-Clavo [25 (link)].
Partial sequences of the 16S rRNA gene obtained from each isolate were assembled into a contig and then compared with the sequences of organisms represented in the EZBioCloud 16S Database (https://www.ezbiocloud.net ) using the “Identify” service [26 (link)]. Species assignments were based on closest hits [27 (link)]. 16S rRNA gene sequences retrieved from the database and related to the unknown organism gene were selected for alignment in the Clustal X program [28 (link)]. Phylogenetic analyses were performed using the Mega version 11.0 program [29 (link)], and the phylogenetic tree was constructed from the evolutionary distances calculated by the neighbour-joining method, with bootstrap values from 1000 resamples.
Partial sequences of the 16S rRNA gene obtained from each isolate were assembled into a contig and then compared with the sequences of organisms represented in the EZBioCloud 16S Database (
4
AAAS Gene Mutation Analysis
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The study, accomplishing the Declaration of Helsinki, was approved by Ethic Committee of the Istituto Auxologico Italiano, and all patients or their tutors gave a written informed consent. Genomic DNA of the probands and their parents was extracted from 3mL of whole blood with Puregene DNA Purification Kit (GENTRA Systems) according to the manufacturer recommendations and stored at -20°C until use. The 16 exons and the flanking intronic regions of the AAAS gene (OMIM 605378) were tested for mutations by sequence analysis. PCR amplification of the 16 AAAS fragments was performed using a specific primer sets (available upon request). All primers were ordered from Eurofins (Ebersberg, Germany).
Each PCR product was checked on a 2% agarose gel TAE 1X and purified with GFX™ PCR DNA/Gel Band Purification Kit (GE Healthcare IL, United States). the PCR product were sequenced using a Big-Dye Terminator v3.1 cycle sequencing kit (Applied Biosystem Waltham, MA, USA) according to the manufacturer's recommendations and after purification with CENTRI-SEP Spin Columns (Princeton Separations, Freehold, NJ, USA) was processed by an ABI-PRISM 3100 automated sequencer (Applied Biosystem Waltham, MA, USA). Sequences with DNA variations were confirmed on a different DNA aliquot.
Each PCR product was checked on a 2% agarose gel TAE 1X and purified with GFX™ PCR DNA/Gel Band Purification Kit (GE Healthcare IL, United States). the PCR product were sequenced using a Big-Dye Terminator v3.1 cycle sequencing kit (Applied Biosystem Waltham, MA, USA) according to the manufacturer's recommendations and after purification with CENTRI-SEP Spin Columns (Princeton Separations, Freehold, NJ, USA) was processed by an ABI-PRISM 3100 automated sequencer (Applied Biosystem Waltham, MA, USA). Sequences with DNA variations were confirmed on a different DNA aliquot.
5
Genotypic Identification of LAB for Cactus Pear Juice Fermentation
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Genotypic identification was carried out in most promising LAB strains for cactus pear juice fermentation. Identification of selected LAB with different RAPD-PCR profiles was carried out by partial 16S rRNA gene sequencing. Genomic DNA was extracted as described by Pospiech and Neumann (1995) . Oligonucleotide primers (PLB16, 5 0 -AGAGTTTGATCCTGGCTCAG-3 0 and MLB16, 5 0 -GGCTGCTGGCACG-TAGTTAG-3 0 ) were used to amplify the variable (V1) region of the 16S ribosomal RNA gene. PCR products were electrophoresed in 1.0% (w/v) agarose gels, stained and visualized as above described. Amplicons were excised from the gel and purified using a GFX PCR DNA gel bandpurification kit (GE Healthcare, UK). Purified PCR products were sequenced at CERELA-CONICET by using an ABI 3130 DNA sequencer (Applied Biosystems, Foster, CA). Taxonomic strain identification was performed by comparing the sequences for each isolate with those reported in the Basic BLAST database (NCBI; National Center for Biotechnology Information) and SeqMatch database (RDP; Ribosomal Database Project, Release 11.2). Strains showing homology of at least 97% were considered to belong to the same species.
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