Procedure for the isolation of DNA from clotted blood of birds

Abstract

In order to isolate genomic DNA economically and safely, from coagulated blood samples, to perform molecular genetic studies, the following protocol was optimized: approximately 250 μL of blood clot were taken, 400 μL of buffer was added cell lysis (10mmol / L Tris-HCl, pH 8.0, 10 mmol / L KCl, 10 mmol / L MgCl₂, 2mmol / L EDTA, pH 8.0; 0.4 mol / L NaCl and 10g / L SDS) and it was left to incubate until the next day at room temperature. 350 μL of chloroform was added and stirred for 10 seconds, then 350 μL of 5M potassium acetate was added, stirred for 10 seconds and centrifuged for 10 minutes (14,000 g). The supernatant was transferred to new tubes and 700 μL of cold isopropanol was added, centrifuged for 5 minutes at 14,000 g and the supernatant discarded. Subsequently, two washes were performed with 70% ethanol (700 μL each), centrifuged for 5 minutes and the liquid was discarded. The sediment was dried 10 min at 45 ⁰C and 8 min at 65 ⁰C and then rehydrated with 20-25 μL TE-1X. The concentration and purity of the DNA isolates were determined by spectrophotometry at 260 nm for an average of 2288.12 ng / μL and 2.04 for the A260/A280 ratio. The suitability of the extracted DNA was evaluated by PCR amplification of the markers MCW0241 and LEI0122. The procedure described allowed to obtain DNA of good quality and in large quantities in a simple, reliable and economical way.