Optimized DNA Extraction from Blood, Tissue, and Sand Flies

DNA was extracted from blood (200 μl) and spleen (25mg) by QIAamp Blood and tissue DNA mini kit (Qiagen, Hilden, Germany) in 60 μl of nuclease free water (Milli-Q) in accordance with the manufacturer’s instructions. From single blood-fed sand flies, DNA was extracted 48 hrs. after xenodiagnosis via Gentra Puregene Tissue DNA Extraction Kit (Qiagen). To achieve maximum yield, tissue digestion was performed overnight with proteinase K in lysis buffer and increased the protein precipitation timing at -20°C for overnight. DNA was eluted in 30 μl of Milli-Q and stored at -20°C for downstream processing. This protocol was optimized for individual sand flies [46 (link)]. The quality of extracted DNA from samples (Blood, tissue and sand flies) was assessed by spectrophotometer (ND-2000 spectrophotometer; Thermo Scientific, USA). DNA samples having 260/280 ratio in range 1.8–2.0 with 260/230 ratio above 1.5 were used for qPCR experiments, which included approximately 95% of samples.