A DNA fragment containing predicted OxyR binding sites in the opvAB regulatory region and labeled with 6-carboxyfluorescein (6-FAM) was prepared by polymerase chain reaction (PCR) amplification using primers FAMGATClargo-5 and FAMGATClargo-3 (Supplementary Table S2). The PCR product was purified with the Wizard® SV Clean-Up System (Promega). The envR control fragment was prepared using primers envR-For-Dnase and envR-Rev-Dnase (26 (link)), and was kindly provided by Elena Espinosa. Thirty five nanogram were used for each reaction. The FAM-labeled probe was incubated at room temperature for 30 min with increasing concentrations of purified 6×His-OxyRC199S in a final volume of 20 μl with 1× OxyR binding buffer [25 mM Tris–HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 5% glycerol, 50 μg/ml bovine serum albumin (BSA), 1 mM DTT, 1 μg/ml poly(dI-dC)]. Protein–DNA complexes were subjected to electrophoresis at 4°C in a 5% non-denaturing polyacrylamide gel in Tris-glycine-ethylenediaminetetraacetic acid (EDTA) buffer (25 mM Tris–HCl pH 7.5, 380 mM glycine, 1.5 mM EDTA). The gel was then analyzed in a FLA-5100 Scanner (Fujifilm, Tokyo, Japan).
Wizard sv clean up system
Manufactured by Promega
Sourced in United States
The Wizard SV Clean-up System is a lab equipment product designed to purify and concentrate DNA samples. It utilizes a silica-based membrane to bind DNA, allowing for the removal of contaminants and salts. The system is intended to provide a simple and efficient method for DNA cleanup prior to downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Dna ladder marker, by Promega (3 mentions)
Sequencher tmv 4.1.4 software for pc, by Gene Codes (2 mentions)
Abiprism 3130 genetic analyzer automated sequencer, by Thermo Fisher Scientific (2 mentions)
Abi prism 3730, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Fla 5100 scanner, by Fujifilm (1 mentions)
Dam methylase, by New England Biolabs (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
7 protocols using wizard sv clean up system
1
OxyR-Mediated Regulation of opvAB
2
In Vitro DNA Methylation and Purification
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PCR fragments were methylated in vitro using Dam methylase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions and subsequently digested with MboI (New England Biolabs). The undigested product was purified using the Wizard® SV Clean-Up system (Promega, Madison, WI, USA).
3
Confirming T. canis Specificity via LAMP
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To confirm the specificity of the LAMP primers, a single round PCR was done based on B3 and F3 outer primers. Amplicons of the ITS2 gene were purified with the Wizard SV Cleanup System (Promega). ABIPRISMTM 3130 Genetic Analyzer automated sequencer (Applied Biosystem, USA) directly sequenced PCR products from 10 randomly selected samples. Contigs (overlapped sequences) from all samples were aligned and edited at consensus positions compared to GenBank sequences of all regional species using Sequencher Tmv.4.1.4 Software for PC (Gene Codes Corporation). T. canis sequences of southwest Iran (Accession nos.; AB743614-AB743617 and AB819327-AB819330) were directly retrieved from GenBank database (FASTA format). The sequences pairwise distances (percent identity and divergence) between sequenced isolates and other country sequences were constructed using the MegAlign program from Laser Gene Bio computing Software Package (DNASTAR, Madison, WI). MEGA 5.05 software with maximum likelihood algorithm and Kimura2-parameter model were used in order to construct phylogeny tree. The diversity (haplotype and nucleotide diversity), neutrality indices (Tajima’s D and Fu’s Fs statistic) and fixation index (Fst) were estimated by DnaSP software version 5.10 [24 ].
4
MLEE and HSP70 Sequencing for Leishmania Identification
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All samples from CLIOC are typed by Multilocus Enzyme Electrophoresis (MLEE) following the internal Standard Operational Procedures (SOPs). Additionally, for the present work, the HSP70 PCR products of the selected strains were obtained and sequenced, as previously reported [11 (link)], as an additional method to identify the species and reveal the polymorphisms within the region analyzed by HRM. The DNA sequencing of the HSP70 genomic region was performed by Fiocruz facilities. Briefly, PCR products were purified with the Wizard SV Clean-up System (Promega, Madison, WI, USA). The final DNA concentration was estimated by comparison with a DNA Ladder Marker (Promega, Madison, WI, USA) in a 1.5% agarose gel. Sequencing was performed with the same primers used for amplification using the BigDyeTerminator v3.1 Cycle Sequencing Kit, and the products were analyzed in an automated DNA sequencer (ABI PRISM-3730—Thermo Fisher Scientific, Waltham, MA, USA). Consensus sequences were generated from forward and reverse strands using the Phred-Phrap-Consed package [34 (link)]. Only sequences with Phred values above 20 were used for contig construction.
5
MLEE and HSP70 Sequencing for Leishmania Identification
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All samples from CLIOC are typed by Multilocus Enzyme Electrophoresis (MLEE) following the internal Standard Operational Procedures (SOPs). Additionally, for the present work, the HSP70 PCR products of the selected strains were obtained and sequenced, as previously reported [11 (link)], as an additional method to identify the species and reveal the polymorphisms within the region analyzed by HRM. The DNA sequencing of the HSP70 genomic region was performed by Fiocruz facilities. Briefly, PCR products were purified with the Wizard SV Clean-up System (Promega, Madison, WI, USA). The final DNA concentration was estimated by comparison with a DNA Ladder Marker (Promega, Madison, WI, USA) in a 1.5% agarose gel. Sequencing was performed with the same primers used for amplification using the BigDyeTerminator v3.1 Cycle Sequencing Kit, and the products were analyzed in an automated DNA sequencer (ABI PRISM-3730—Thermo Fisher Scientific, Waltham, MA, USA). Consensus sequences were generated from forward and reverse strands using the Phred-Phrap-Consed package [34 (link)]. Only sequences with Phred values above 20 were used for contig construction.
6
Genetic Diversity Analysis of cox1 Gene
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PCR products were purified with the Wizard SV Clean-up System (Promega). The final DNA concentration was estimated by comparison with a DNA Ladder Marker (Promega) in 2 % agarosegel. All amplicons were directly sequenced by targeting cox1 gene in both directions using the mentioned primers by ABIPRISMTM 3130 Genetic Analyzer automated sequencer (Applied Biosystem, USA). Ambiguous (heterozygous) sites were coded using the standard IUPAC codes for combinations of two or more bases. Contigs from all samples were aligned, justified and edited in consensus positions compared to GenBank sequences of all regional species using Sequencher Tmv.4.1.4 Software for PC (Gene Codes Corporation). The diversity testes of analyzed sequences (Haplotype diversity; Hd and Nucleotide diversity: Pi) were determined by DnaSP 5.10.1 software [49 (link)].
7
Extraction and Amplification of Satellite DNA
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Genomic DNA was extracted from young leaves using double precipitation with cetyltrimethylammonium bromide (CTAB) according to Grattapaglia and Sederoff (1994) . DNA concentrations were determined by spectrophotometry.
From the alignment of the putative monomers of the satDNA "ATR-2", a pair of specific primers was designed based on the most conserved regions of the motifs. Polymerase chain reaction (PCR) was performed in a final volume of 10 µl in the presence of 50 ng of total genomic DNA, 2 mM each primer, 0.2 mM dNTPs, 2.5 mM MgCl 2 , and 0.5 U of Taq polymerase in the corresponding PCR buffer. After an initial denaturation step at 94 °C for 5 min, amplifications were conducted for 35 cycles at 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 1 min, with a final elongation step at 72 °C for 5 min. The PCR products were analyzed by electrophoresis, and the bands were cut out of the gel, purified using the Wizard ® SV Cleanup System (Promega), and ligated to pGEM ® -T Easy Vector (Promega). The recombinant clones were sequenced by Macrogen Inc. Service (Korea).
From the alignment of the putative monomers of the satDNA "ATR-2", a pair of specific primers was designed based on the most conserved regions of the motifs. Polymerase chain reaction (PCR) was performed in a final volume of 10 µl in the presence of 50 ng of total genomic DNA, 2 mM each primer, 0.2 mM dNTPs, 2.5 mM MgCl 2 , and 0.5 U of Taq polymerase in the corresponding PCR buffer. After an initial denaturation step at 94 °C for 5 min, amplifications were conducted for 35 cycles at 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 1 min, with a final elongation step at 72 °C for 5 min. The PCR products were analyzed by electrophoresis, and the bands were cut out of the gel, purified using the Wizard ® SV Cleanup System (Promega), and ligated to pGEM ® -T Easy Vector (Promega). The recombinant clones were sequenced by Macrogen Inc. Service (Korea).
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