Optimization of DNA extraction protocol for insects in the order Odonata

RIPPEL CAMILA; SAWOSTJANIK SILVANA; AYALA, MAHIA; WALANTUS, LEONARDO HORACIO

La Plata, Buenos Aires

Congreso; International Congress of Odonatology; 2015

Instituto de Limnología ?Dr. R.A. Ringuelet"

Obtaining good quality, unpolluted DNA is essential for the application of any molecular technique. In contrast to other insects, there are no published DNA extraction protocols for dragonflies that are both efficient and inexpensive. The objective of this study was to obtain a DNA extraction protocol for insects in the order Odonata. The insects were collected in the adult stage, in fortnightly samples from July to November 2014, in the city of Posadas, Misiones. Taxonomic identification was performed according to the keys proposed by Garrisson et al. (2006-2010). The study was carried out with the species Oxyagrion sp. and Argentagrion ambiguum from the family Coenagrionidae (Odonata: Zygoptera), and Oligoclada sp. from the family Libellulidae (Odonata: Anisoptera), because these were the most abundant. The thoracic muscle was used for the insulation of the whole genetic material. A modified DNA extraction protocol for mosquitoes proposed by Gaillard & Strauss (1990) was used. First, the tissue was macerated in 500 μl of extraction buffer (0.5 M EDTA; 1M Tris-HCl pH 8.25; 2% SDS, 1M Saccharose) and 5 μl proteinase K. Then it was incubated in dry bath for 20 minutes at 65 °C. 120 μl of 4M Potassium Acetate was added for protein precipitation.The homogenized was placed on ice for 10 minutes and centrifuged at 12000 rpm for 10 minutes. After recovering the supernatant which contained DNA, 35μl of 4M Sodium Acetate and 1000 μl of absolute ethanol were added, and it was then incubated for 10 minutes at room temperature with the purpose of getting a good precipitation of the DNA. Next, it was centrifuged for 20 minutes at 12000 rpm. The obtained pellet was dried in a thermostated bath for 20 minutes at 37 °C and resuspended in 50 μl ddH2O. In order to verify the integrity of the extracted nucleic acid, a 1% agarose gel electrophoresis was performed. It was possible to obtain good quality DNA that was amplifiable by using proposed primers for ?Barcode?. Being able to draw on techniques that do not compromise the safety of lab personnel and are also simple and inexpensive constitutes and advantage for the evolution of research methodologies.