A total of 1 μl amplicon was mixed with 8.8 μl of deionized formamide (Applied Biosystems, Foster City, CA, USA) and 0.2 μl of GeneScan 500 ROX Size-standard (Applied Biosystems). The mixed samples were denatured at 95 °C for 5 min, followed by immediate cooling at 4 °C for 3 min. The cooled samples were injected into capillaries by applying a voltage of 15 kV for 5 s. Electrophoresis was performed for 1 h at 35 °C and a voltage of 15 kV. CE-SSCP analysis was conducted on the ABI3130xl instrument (Applied Biosystems) with 16 × 50 cm capillary arrays (Applied Biosystems). The capillary arrays were filled with 15 wt% PEO-PPO-PEO triblock copolymer (Pluronic F108; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.7 × reaction buffer (Applied Biosystems).
Abi3130xl instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI3130xl is a genetic analysis instrument manufactured by Thermo Fisher Scientific. It is designed for DNA sequencing and fragment analysis applications. The ABI3130xl utilizes capillary electrophoresis technology to separate and detect DNA molecules. The instrument features four capillaries and multiple-color fluorescence detection capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Deionized formamide, by Thermo Fisher Scientific (1 mentions)
Genescan 500 rox size standard, by Thermo Fisher Scientific (1 mentions)
Truseq custom amplicon kit, by Illumina (1 mentions)
Maxwell 16 dna purification kit, by Promega (1 mentions)
Applied biosystems sanger sequencing kit, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
P gemt easy vector a1360, by Promega (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
7 protocols using abi3130xl instrument
1
Capillary Electrophoresis SSCP Analysis
2
Targeted TP53 Sequencing in PT-res Cells
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For TP53 sequencing of PT-res pool cells, genomic DNA was extracted from cultured cells using Maxwell 16 DNA purification kit (Promega). Then, 50 ng of genomic DNA was amplified with TruSeq Custom Amplicon kit (TSCA, Illumina, San Diego, CA, USA) specially designed for the targeted sequencing of TP53 (13 amplicons) among others. Libraries were run in an Illumina MiSeq instrument achieving a median coverage >1000 reads. Data were aligned to human reference genome hg18 and analyzed, after quality control, using Variant Studio and the IGV program, reporting only variants with a mutant allelic frequency (MAF) greater than 5%. We considered not only variants inside the coding sequence but also the ones inside the 5′-, 3′-untranslated regions (UTRs) and inside splicing regulatory elements [17 (link)] (http://p53.iarc.fr/TP53GeneVariations.aspx ). Validation of NGS data was performed using the DNA Sanger sequencing method using the Applied Biosystems™ Sanger Sequencing Kit and the ABI3130xl instrument (Applied Biosystems, Waltham, MA, USA).
3
MLVA-8 Genotyping of Bacillus anthracis
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MLVA-8 genotyping was performed as described by Keim et al. (24 (link)) and Sue et al. (25 (link)). Briefly, six chromosomal loci (vrrA, vrrB1, vrrB2, vrrC1, vrrC2, and CG3) and two plasmid loci (pXO1-aat and pXO2-at) were amplified by PCR and the resulting DNA fragments were separated on an ABI 3130xl instrument or an ABI 3500xl instrument (Applied Biosystems, by Thermo Fisher Scientific, Waltham, MA, USA).
4
Genomic DNA Extraction and Molecular Identification of Sporothrix spp.
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Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) from 500 mg of mycelial tissue as input material using a glass bead protocol with a Precellys 24 instrument (Bertin Instruments, Montigny-le-Bretonneux, France). DNA integrity was assessed by running 5 μL aliquot of the extraction on a 0.8% agar agarose-gel electrophoresis stained with 0.5 µg/ml ethidium bromide. To estimate the DNA yield we used a NanoDrop 1000c Instrument (Thermo Fisher Scientific, Waltham, MA, USA). We then amplified a segment of the calmodulin gene by PCR and Sanger sequenced it in an ABI 3130xl Instrument (Applied Biosystems). PCR setting and cycling conditions were done as previously described [6 (link)]. We performed initial nucleotide BLAST analysis (BLASTn) for each amplicon to retrieve the best hit results for each strain on the NCBI database. All extractions (N=11) showed Sporothrix spp. as the closest BLAST match.
5
Characterization of Hemp Subtelomeric Repeats
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A search for the hemp subtelomeric repeat was performed with a BLAST search (http://blast.ncbi.nlm.nih.gov ) using the hop subtelomeric repeat sequence (GU831574) to probe the Cannabis sativa whole-genome shotgun contigs. In the search results, a contig (AGQN01004814) with one repeat of low homology to the hop subtelomeric tandem repeat was found. Nineteen tandemly repeated units about 375 bp in size from this contig were found using the GenDoc software (http://gendiapo.sourceforge.net ) and were subsequently used for primer design. The primers (CS-1f 3′-GGTACCACTATGAGAAATGTGAGA-5′ and CS-1r 3′-CCTTTGTGAAATGTGGCCC-5′ ) used to amplify the detected repeat units were designed using PRIMER3 v.0.4.0 (http://frodo.wi.mit.edu ). The PCR products from the CS-1 reaction were purified and cloned into the pGEM®-T Easy Vector A1360 (Promega, USA) in E. coli cells. Plasmids with inserts were isolated from cells using the GeneJET ™ Plasmid Miniprep Kit (Fermentas, Lithuania). The nucleotide sequences of the inserts were determined on an ABI3130XL instrument (Applied Biosystems, Inc., USA) after sequencing reactions were performed with a Big Dye Terminator v 1.1. Cycle Sequencing Kit (Applied Biosystems, Inc., USA).
6
Viral RNA Extraction and Sequencing
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Viral RNA was extracted using MagNA pure TNA high throughput kit (Roche). NPS samples were screened for respiratory viruses using multiplex real-time reverse-transcription PCR (rRT-PCR) [32 (link)]. A sample was determined positive if rRT-PCR cycle threshold (Ct) value was <40.0 [33 (link)]. Ct value was assumed to be related to viral load. VP4/VP2 sequencing (approximately 450 bp) was used for species and type assignment. Primers amplifying approximately 549 bp of the VP4/VP2 region (F1: 5′-CCGGCCCCTGAATGYGGCTAA-3′, F2: 5′-ACCRACTACTTTGGGTGTCCGTG-3′; R1: 5′-TCWGG HARYTTCCAMCACCANCC-3′, R2: 5′- ACATRTTYTSN CCAAANAYDCCCAT-3′) were used in a nested 25-μL reaction [34 (link)]. PCR products were purified (QIAquick PCR purification Kit, Qiagen) and sequenced on an ABI 3130xl instrument (Applied Biosystems).
7
DNA Sequencing and BLAST Analysis
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DNA sequencing was performed at PT Genetika Science, Jakarta and 1st Base, Malaysia, using Big Dye Terminator v.3.1. and a DNA sequencer (ABI 3130XL instrument, Applied Biosystems, Carlsbad, California, USA). Database searches were carried out using the internet through the Basic Local Alignment Search Tool (BLAST) database tracking program in the GenBank database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). The 16S rRNA option was selected on the BLAST nucleotide selection.
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