Wizard sv gel kit

RNA was extracted with the use of the Trizol (Invitrogen) system following the manufacturer's instructions and RNA samples were submitted to Dnase to remove traces of DNA. The PCR-select cDNA subtraction kit (Clontech) was used in order to obtain differentially expressed transcripts [17 (link)] according to the manufacturer's instructions. Subtractions were carried out from EGG and 452 samples. Fragments of cDNA subtracted were extracted from gel, purified by Wizard SV gel kit and PCR clean-up system (Promega), and then cloned into pGEM-T easy vector (Promega) according to the manufacturer's instructions. Cloning was carried out using Escherichia coli bacteria DH5α (Life Technologies). Individual colonies were grown in 100 μL LB-ampicillin at 37°C, plasmid was isolated, and the presence of the insert was confirmed by digestion reaction using EcoRI (5 U) followed by a PCR using nested primers (Clontech). For those clones presenting insert, plasmidial DNA was isolated from bacterial cultures using Wizard plus SV minipreps (Promega) kit. Positive clones were sequenced in accordance with a method previously described [18 (link)], making use of DYEnamic ET dye terminator kit (MegaBACE, GE Healthcare).

Primers were designed based on the sequence of a non-redundant PilV protein (AWP39907.1; GenBank, ncbi.nlm.nih.gov/genbank) of A. thiooxidans ATCC 19377 (NZ_SZUV01000001.1:273124-274257; GenBank), using Vector NTI Express (Thermo Fisher Scientific). The primers were then synthesized by T4 Oligo (México) as follows: pilV forward: 5'-CTCACTCTCATTGACTATGATCG-3'; pilV reverse, 5'-TCAGTATCCCACGATGGTTTG- 3'. We obtained the PCR-amplified region of 444 bp of pilV.
The pilV mRNA was amplified using GoTaq DNA Polymerase (Promega, USA) according to the protocol provided by the manufacturer. The PCR cycling conditions were: Initial denaturation (95°C, 1 min), 35 cycles consisting of denaturation (95°C, 1 min), primer annealing (55°C, 1 min), and extension (72°C, 1:20 min); followed by a final extension step (72°C, 5 min). PCR products were analyzed by electrophoresis in 1% (w/v) agarose gels (Sigma) and purified by gel extraction with the Wizard SV Gel kit and PCR Clean-Up system (Promega).

The amplification product of ORF2 was purified using reagents and protocols of the commercial Wizard SV Gel kit and PCR Cleaning System (Promega, USA). Sequencing reactions were performed using reagents and protocols of Big Dye Terminator 3.1 kit (Applied Biosystems, EUA). Phylogenetic analyses were conducted with Bayesian inference using Markov Chain Monte Carlo (MCMC) statistical framework implemented in the program BEAST v1.8.1 [30 (link)] under TRN+G nucleotide substitution model. A phylogenetic tree, based on the HEV ORF2 region (302bp), was constructed with sequences retrieved from GenBank, including prototype sequences from HEV genotypes 3 and 4.

The ITS1-rDNA was amplified by conventional PCR using the primers L5.8S: 5′-TGATACCACTTATCGCACTT-3′ and LITSR: 5′-CTGGATCATTTTCCGATG-3′ [14 (link)]. Amplification reactions were performed in volumes of 50 μL. Amplicons from the PCR-positive samples (300–350 bp, depending on the species) were visualized on a 2% agarose gel and purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega, Madison, USA). The products were then sequenced with the same primers used in the PCR assay. Sequencing was performed on an automated sequencer at Plataforma de Sequenciamento Genômico ABI-3730 (Oswaldo Cruz Institute/FIOCRUZ).
Sequence alignment was performed using SeqMan Pro (DNASTAR) and comparisons were conducted with Leishmania reference strains sequences obtained from the GenBank database. Phylogenetics analyses with the evolutionary history was inferred using the maximum likelihood method based on the Jukes–Cantor model, and the sequences were aligned using Molecular Evolutionary Genetic Analysis (MEGA) version 6. This same software was used to calculate a distance matrix and the genetic distance percentage between the test samples and reference strains of Leishmania spp.

Sanger sequencing of the internal transcribed spacer of ribosomal DNA (ITS1-rDNA) was performed only in one case where it was not possible to obtain taxonomic identification through MLEE or PCR-RFLP.
The ITS1-rDNA was amplified by conventional PCR using the primers L5.8S: 50-TGATACCACTTATCGCACTT-30 and LITSR: 50-CTGGATCATTTTCCGATG-30. Amplification reaction was performed in volume of 50 μL. Amplicons from the PCR positive sample were visualized on 2% agarose gel and purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega, Madison, Wisconsin, USA). The products were then sequenced with the same primers used in the PCR assay. Sequencing was performed on an automated sequencer at Plataforma de Sequenciamento Genômico ABI-3730 (Oswaldo Cruz Institute/Fiocruz).
Sequence alignment was performed using SeqMan Pro (DNASTAR, Madison, Wisconsin, USA) and comparisons were conducted with Leishmania reference strains sequences obtained from the GenBank database. Phylogenetics analyses with the evolutionary history were inferred using the maximum likelihood method based on the Jukes-Cantor model and the sequences were aligned using Molecular Evolutionary Genetic Analysis (MEGA) version 6 (Tokyo Metropolitan University, Tokyo, Japan; Arizona State University, Arizona, USA; King Abdulaziz University, Jeddah, Saudi Arabia).

The sequences obtained from both inserts (Pe69Q4G9 and Pe85Q4F4) were aligned using ClustalX software (Larkin et al., 2007 (link)) to obtain a single contig. Specific primers were designed at the sequence ends of this contig in order to find out whether the circular chloroplast genome was complete. PCR reactions were then performed using a 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) in reaction mixtures containing 20 ng template DNA (P. edulis accession ‘IAPAR-123’), 1× buffer, 1 mM MgCl₂, 0.2 mM of each dNTP, 0.3 μM of the forward and reverse primers, 1.2 U Go Taq Flex DNA polymerase (Promega, Madison, WI, USA), and ultra-pure water to bring the final volume up to 20 μL. The thermal profile for amplification was: 95°C for 5 min, 35 cycles at 95°C for 40 s, 55°C for 40 s and 72°C for 1 min, followed by a final 8 min incubation at 72°C. The amplified fragments were checked on 1% (w/v) agarose gel with a 100 bp molecular size standard Invitrogen (Carlsbad, CA, USA). The PCR product was purified using the Wizard^® SV Gel kit and PCR Clean-Up System (Promega), and then used as a template for the sequencing reaction based on the Sanger method. It was subjected to capillary electrophoresis in the ABI Prism 3100 sequencer (Applied Biosystems). The sequence of the PCR product was aligned with the single contig to obtain the complete sequence of the cp genome.

Penicillin, streptomycin, Schneider's Drosophila medium, cell culture flasks, and the fluorescent stain Nancy-520 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Fetal calf serum (FCS), primers for proteases, kDNA3 and housekeeping genes, TRIzol reagent, RNase H enzyme, DEPC-treated water, deoxyribonucleotide phosphate solution (dNTPs), Platinum Taq DNA Polymerase (DNA Polymerase), and Taq Platinum PCR buffer were purchased from Invitrogen, Life Technologies (USA). iScript cDNA Synthesis kit was purchased from Bio-Rad Laboratories, (Hercules, CA). GoTaq® qPCR Master Mix, Wizard SV Gel Kit, and PCR Clean-UpSystem were purchased from Promega Corporation (USA). High Pure PCR Template Preparation Kit was purchased from Roche Molecular Systems, Inc. Chloroform and ethanol were purchased from Merck (Brazil). All reagents were of analytical grade or higher.