Optimizing factors influencing DNA extraction from fresh whole avian blood

A study was conducted to optimize the efficient combination of lysis buffer, Proteinase K, incubation time, phenol-chloroform isoamyl alcohol (PCI) volume, spinning speed (rpm), and precipitation agent on quantity and quality of DNA extracted form whole avian blood. Whole blood samples were collected in EDTA and quickly transformed to laboratory for DNA extraction quickly. The lysis buffer used had the composition of 5M NaCl, 1M Tris (pH=8.0), 0.5M EDTA and 20% SDS. The effect of different levels for each above-mentioned factor has been examined using General Linear Models or t-test procedures of SAS® software. The volume which was removed from top aqueous part after first and second PCI washings were included in the models as a co-variant. The variables of interest were consisted of OD280, OD260, OD260/OD280 (as quality criterion), total extracted DNA, extraction efficiency (μg DNA/μl blood), assay scores for easiness of removing the top aqueous phase after first (assay 1) and second (assay 2) spinning. Optimum level of each factor concerned for DNA extraction from whole fresh avian blood was found to be lysis buffer: blood sample ratio = 31-36(μl: μl), incubation time=60-70 min at 59-60oC, two times washing with PCI at 1.2-1.3 PCI: top aqueous phase (μl: μl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 10000 rpm for 10-20 min, precipitation of DNA with 1.5 –2.0 volume absolute ethanol.

کلیدواژه ها:

DNA extraction ، avian ، whole blood