A new DNA extraction protocol for screwworm fly Cochliomyia species (Diptera: Calliphoridae)

Abstract

Modifications to the DNA isolation protocol from Cochliomyia spp., are suggested based on the Chelex® 100 reactive. To apply the sterile insect technique (SIT) program it is necessary to study the molecular variations of endemic populations with efficient, fast and low costs techniques. The test samples were collected in the Pichincha province of Ecuador. The isolation protocol had 3 steps: (a) pretreatment (optional), (b) mechanic and chemical lysis, (c) two incubations; then the supernatant were separated by centrifugation. Furthermore, variations in concentrations of magnesium chloride present in the master mix were evaluated. Results showed a high efficiency in isolation with approximately 1.20 h of manipulation (without pretreatment). Additionally, the quality of the amplicon that was visualized on 2% agarose (w/v) showed that the magnesium chloride concentration was influential in the PCR reaction mix.

Eco-epidemiological aspects, natural detection and molecular identification of Leishmania spp. in Lutzomyia reburra, Lutzomyia barrettoi majuscula and Lutzomyia trapidoi

“…Para la extracción del ADN se siguió el protocolo de Echeverría, et al (30). Las modificaciones consistieron en agregar 20 µl de proteinasa K en 100 µl de la solución tampón de extracción para incubar cada muestra durante una hora a 60 °C y a 1.300 rpm, y luego a 100 °C …”

Transinfection of buffalo flies (Haematobia irritans exigua) with Wolbachia and effect on host biology

“…A Chelex extraction protocol modified from Echeverria-Fonseca et al [51] was used for extraction of DNA from the embryonic and adult microinjected samples. Briefly, flies were homogenised using a Mini-Beadbeater (Biospec Products, Bartlesville, Oklahoma, USA) for 5 min in 2 ml screw-cap vials with 2 g of glass beads (2 mm) and 200 µl of buffer containing 1× TE buffer and 10% Chelex ® -100 (Bio-Rad Laboratories, CA, USA).…”

Fatores Físicos E Atributos Florais Afetam a Produção De Néctar?

“…(5' -GGTAAAATTAAAATATAAACTTC-3') (ECHEVERRÍA-FONSECA et al, 2015), sendo realizada variações nas quantidades de reagentes para a amplificação. As reações foram preparadas em volume total de 50µL, sendo, 5µL de Tampão 10X, 3µL de MgCl2 (50mM), 1µL de dNTP's (10mM), 1 µL de primers forward e reverse (10 moles), 1,3µL de Taq polimerase (5U/µL), 1,5 ng/µL de DNA diluído a 100 ng/ µL e 36,2 µL de água ultrapura.…”