Chromosomal DNA was extracted from the B. cereus vegetative and sporulating cells, according to the method described previously11 (link). The junction sequences at the attB (composite gerE) and attG (the excised gin element) sites were amplified from 100 ng of the chromosomal DNA with the primer sets PA246/PA249 and PA247/PA248, respectively. PCR products were separated by agarose gel electrophoresis, and stained using EZ-Vision DNA Dye (AMRESCO, OH, USA). Gel images were cropped, using Paintgraphic2 (SOURCENEXT, Tokyo, Japan). DNA sequencing was performed, using an ABI 3500 DNA analyzer (Thermo Fisher Scientific, WI, USA) with the PA246 (for the 5′- and composite gerE) or PA248 (for 3′-gerE and the excised gin element) primers and the BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).
Abi 3500 dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 3500 DNA analyzer is a capillary electrophoresis-based system used for DNA sequencing and fragment analysis. It employs multi-color fluorescence detection technology to analyze DNA samples. The instrument is designed to deliver accurate and reliable results for a variety of genetic analysis applications.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Big dye v3.1 terminator kit, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp, by Beckman Coulter (2 mentions)
Ez vision dna dye, by Avantor (1 mentions)
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Genemarker, by SoftGenetics (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Dna mini kit, by Qiagen (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
13 protocols using abi 3500 dna analyzer
1
Chromosomal DNA Extraction and Sequence Analysis
2
Sanger Sequencing Variant Confirmation
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Sanger sequencing was performed for confirmation of all variants, to fill the
regions missed by the custom panel design and low-coverage regions, and for the
analysis of clinical utility. gDNA was amplified using specific primers designed
for the free software Primer3 v.0.4.0. (available upon request). Amplicons were
sequenced by both ends using the Big Dye Terminator v3.1 cycle sequencing kit
(Thermo Fisher Scientific), and fragments were resolved on an ABI 3500 DNA
Analyzer (Thermo Fisher Scientific). Analysis of results was performed with the
BioEdit v7.2.5 free software.
regions missed by the custom panel design and low-coverage regions, and for the
analysis of clinical utility. gDNA was amplified using specific primers designed
for the free software Primer3 v.0.4.0. (available upon request). Amplicons were
sequenced by both ends using the Big Dye Terminator v3.1 cycle sequencing kit
(Thermo Fisher Scientific), and fragments were resolved on an ABI 3500 DNA
Analyzer (Thermo Fisher Scientific). Analysis of results was performed with the
BioEdit v7.2.5 free software.
3
Characterization of attP site by PCR
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PCR for a 2231 bp DNA fragment harboring the attP site was performed using the chromosomal DNA of the B. subtilis 168 sporulating cells and the P100/P108 primers. Two microgram of the PCR product were mixed with 0.3 μM SprA in 100 μl of the reaction solution containing 10 mM Tris–HCl (pH 8.0), 20 mM NaCl and 0.1 mM DTT. The reaction mixture was incubated at 37°C for 12 h in the presence of 30% ethylene glycol and 5% glycerol to accumulate the cleavage products (54 (link)). The cleavage products were treated by proteinase K, extracted by phenol, and precipitated using 100% ethanol. DNA pellets were dissolved in TE buffer, gel purified, and directly sequenced using the P107 (for the left half-site of attP) or P109 (for the right half-site) primers, a BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, WI, USA) and an ABI 3500 DNA analyzer (Thermo Fisher Scientific).
4
Genomic DNA Extraction and NLGN3 Gene Sequencing
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Genomic DNA was extracted from peripheral blood using a DNA extraction kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany) as per the manufacturer’s instructions. All seven exonic regions of the NLGN3 gene were amplified by polymerase chain reaction (PCR) using PCR reaction kits (New England Biolabs, Ipswich, MA, USA) with specifically designed primers. Sequencing of the amplified product was performed on an ABI 3500 DNA analyzer using a Big Dye terminator version 3.1 cycle sequencing kit (Applied Biosystems, Waltham, MA, USA). Sequencing results were analyzed on DNA Sequencing Analysis Software v5.4 (Applied Biosystems).
5
Amplified Fragment Length Polymorphism Analysis
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AFLP reactions were performed following Sun et al. [2 (link)] with a few minor modifications. Six informative and reliable pairs of MseI (M-GACGATGAGTCCTGAG ) and EcoRI (E-CTCGTAGACTGCGTACC ) primers used in amplification were selected from a set of 100 primer pairs. The amplification PCR reaction was performed in a final volume of 20 μL as Zhang et al. [39 ]. The capillary electrophoresis was performed using ABI 3500 DNA analyzer (Applied Biosystems, USA) and those fragments were analyzed using GeneMarker® (version 2.6, SoftGenetics, LLC).
6
Sanger Sequencing with BigDye Terminator
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Sequencing was done following the kit’s protocol (BigDyeTerminator v3.1, Applied Biosystems). The same PCR primers have been used for sequencing. The reaction has done in An ABI-3500 DNA-Analyzer (Applied Biosystems).
7
Comprehensive FLT3 and NPM Mutation Analysis
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DNA was extracted from BM/PB samples using the Qiagen DNA Mini kit (Qiagen, Hilden, Germany). The ITD (Internal tandem duplications) and tyrosine kinase domain (TKD) containing regions of the FLT3 and exon 12 of the NPM genes were amplified using fluorescent labeled primers. The size of the ITD/NPM PCR product was determined by ABI 3500 DNA analyzer (Applied Biosystems, Foster City, California, USA). The TKD PCR product was digested with EcoRV, and the presence of the mutation was determined on agarose gel electrophoresis. RNA was extracted from WBCs using Trizol reagent, complementary DNA was then synthesized using reverse transcriptase, followed by nested RT-PCR (PML-RARA) using Roche First Start Taq polymerase and the products were separated by agarose gel electrophoresis.6 (link)
8
Confirming C. pseudotuberculosis PCR Results
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In order to confirm the quadruplex PCR results, ten randomly chosen isolates were tested further in singleplex PCR assays with the four C. pseudotuberculosis-specific primer pairs. PCR products were purified using Agencourt AMPure XP (Beckman Coulter Company, Beverly, Massachusetts, USA) according to the manufacturer’s instructions, and each product was sequenced in both directions using primers targeting the 16S rRNA, rpoB, pld and narG gene and the Big Dye V3.1 Terminator Kit (Applied Biosystems, USA) using an ABI 3500 DNA analyzer (Applied Biosystems, California, USA). Sequences were analyzed on the Geneious suite of molecular biology (http://www.geneious.com ) with 16S rRNA (GenBank accession nos X81916, X81907, and X84255), rpoB (GenBank accession no. AY492239), pld (GenBank accession nos L16586 and L16587) and narG (GenBank accession no AJF93840.1) as the reference genes.
9
Canine Parvovirus Detection and Sequencing
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All stool samples were submitted to total DNA extraction with Invisorb Spin Tissue Mini Kit (Invitek, EUA). Amplification was performed as previously described by Ref. [1] (link) with primers 555F-5′CAG GAA GAT ATC CAG AAG GA3′ and 555R-5′GGT GCT AGT TGA TAT GTA ATA AACA3′, resulting in a fragment of 583 bp from VP2. Aliquots of stool samples positive for CPV by PCR (n = 82) were further submitted to C. difficile and C. perfringens detection, as described next. In addition, 20 random CPV positive samples were selected for VP2 sequencing, as described previously [1] (link). Briefly, PCR products were purified using Agencourt AMPure XP (Beckman Coulter Company, Beverly, Massachusetts, USA) according to the manufacturer's instructions, and each product was sequenced in both directions using primers the 554F and 555R, and the Big Dye V3.1 Terminator Kit (Applied Biosystems, USA) using an ABI 3500 DNA analyzer (Applied Biosystems, California, USA). In order to prevent detection of canine CPV post-vaccination shedding, dogs without vaccination status record, or vaccinated less than 3 weeks before the analysis were excluded from the study. Also, all dogs simultaneously positive for CPV and C. perfringens or C. difficile infection were selected for VP2 sequencing.
10
Comprehensive SARS-CoV-2 Screening Protocol
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CoV screening of pooled or individual samples was performed by amplifying a 440-bp fragment of the RdRp gene of all the α-CoVs and β-CoVs using conserved primer pairs as described previously [7 (link),60–62 (link)]. Specific primers were designed from assembled contigs of sarbecovirus and their closest reference sequence identified by BLAST from Genbank. Each ORF in the viral genome was amplified with nested specific primers whose PCR product was nearly 1500 bp, and then sequenced with ABI3500 DNA analyzer (Applied Biosystems, USA). PCR products with low concentration, or that were generating heterogeneity in the sequencing chromatograms, were cloned into pMD-18T Vector (Takara) for sequencing.
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