A housekeeping gene, β-actin gene, was used to evaluate the quality of the extracted DNA. The PCR proceeded with total volume of 18.1 ml comprising 12.5 ml Top Taq (Qiagen, Germany), 1.6 ml 25 mM MgCl7 (Promega, Madison, WI), 0.5 ml of each of 20 pmol forward and reverse primers (Table 1 ), and 3.0 ml of extracted DNA. Conditions for the thermal cycling were as follows: initial denaturation of protein for 5 min at 94° C, 35 cycles of denaturation for 30 sec at 94° C, annealing for 30 sec at 55° C, extension for 30 sec for 72° C, and final extension step for 5 min at 72° C to complete the reaction. The expected amplicon size of this gene is 227 base pairs [13 ]. Only the samples that are positive for the β-actin gene were used for the next procedures.
Top taq
Manufactured by Qiagen
Sourced in Germany
Top Taq is a thermostable DNA polymerase enzyme used for amplifying DNA sequences in polymerase chain reaction (PCR) applications. It exhibits high thermal stability and robust performance for reliable DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Mgcl7, by Promega (1 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (1 mentions)
Ribozol rna extraction reagent, by Avantor (1 mentions)
Omniscript reverse transcriptase, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bigdye terminator 3.1 kit, by Thermo Fisher Scientific (1 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
3 protocols using top taq
1
Evaluating DNA Quality via β-Actin Gene PCR
2
Comprehensive RNA Extraction and Analysis
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Total RNA from serum-starved cells was extracted using TRIzol (Life Technologies) or RiboZol™ RNA Extraction Reagent (Amresco, Solon, OH), following the manufacturer’s protocols. The RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and quantitation was performed using a Nanodrop spectrometer (Thermo Fisher). Reverse transcription was performed on DNase I-treated RNA (Amplification grade, Life Technologies) with Omniscript Reverse Transcriptase (Qiagen). Semi-quantitative PCR reactions were carried out using TOPTaq (Qiagen), according to the manufacturer’s protocol. Resulting PCR products were then analyzed on a 2% agarose gel. Quantitative real-time PCR analyses were performed by the RNomics Platform at the Université de Sherbrooke (Sherbrooke, QC, Canada). The sequence of the primers used is listed in Additional file 2 .
3
Malpighiaceae Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted using the 2 × CTAB protocol, modified from Doyle and Doyle [66 ]. Three DNA regions (nuclear PHYC gene, and the ribosomal external and internal transcribed spacers (ETS and ITS)) were selected based on their variability in previous Malpighiaceae studies [2 (link),5 (link),6 (link),8 (link),67 (link)]. Protocols to amplify and sequence ETS and ITS followed Almeida et al. [8 (link)]. For amplification, we used the TopTaq (Qiagen) mix following the manufacturer’s standard protocol, with the addition of betaine (1.0 M final concentration) and 2% DMSO for the ETS region. PCR products were purified using PEG (polyethylene glycol) 11% and sequenced directly with the same primers used for PCR amplification. Sequence electropherograms were produced on an automatic sequencer (ABI 3130XL genetic analyzer) using the Big Dye Terminator 3.1 kit (Applied Biosystems). Additional sequences for PHYC were retrieved from GenBank (Table 3 ). Newly generated sequences were edited using the Geneious software [68 (link)], and all datasets were aligned using Muscle [69 ], with subsequent adjustments in the preliminary matrices made by eye. The complete data matrices are available at TreeBase (accession number S21218). This study was authorized by the Genetic Heritage and Associated Traditional Knowledge Management National System of Brazil (SISGEN #A3B8F19).
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