Adjustment of protocol for the extraction of cowpea DNA.
2025
NORBERTO, R. L. de S. | ZANETTI, G. T. | PINTO, J. M. A. | MENEZES JUNIOR, J. A. de | HOOGERHEIDE, E. S. S. | RAYANE LUCAS DE SOUZA NORBERTO, UNIVERSIDADE FEDERAL DE MATO GROSSO; GÉSSICA TAIS ZANETTI, UNIVERSIDADE DO ESTADO DE MATO GROSSO; JOYCE MENDES ANDRADE PINTO, CPPSE; JOSE ANGELO NOGUEIRA DE MENEZES JUNIOR, CNPMS; EULALIA SOLER SOBREIRA HOOGERHEIDE, CPAO.
The use of DNA extraction protocols provides genetic material with high quality, quantity, stability and purity. Thus, the objective of this study was to adjust the protocol for DNA extraction of leaf tissue from cowpea (Vigna unguiculata). Adjustments were made to the DNA extraction protocol using CTAB (cetyltrimethylammonium bromide). The variables tested were CTAB (2%, 3%, 4% and 5%), β-mercaptoethanol (1% and 1.5%), Polyvinylpyrrolidone (2% and 3%), mechanical maceration of plant tissue for 30 s, 1 min and 2 min, and manual maceration (with a pistil and glass rod); liquid nitrogen and incubation in a water bath (30 min and 1 h). The results show that the 5% concentration of CTAB was more efficient, and it is thus recommended for DNA extractions of this species. The different concentrations of β-mercaptoethanol did not interfere with the result. Mechanical maceration using TissueLyser II at 1 min was efficient for good quality DNA extraction. The use of liquid nitrogen in the maceration was essential for the integrity of the genetic material. The reduction in water bath time resulted in time savings though did not affect DNA quality. The ISSR primer evaluated amplified regions of the genome and proved the efficiency of the extraction.
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