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Optimizing factors influencing DNA extraction from fresh whole avian blood
Abstract
A study was conducted to optimize the efficient combination of lysis buffer, proteinase K, incubation time, phenol-chloroform-isoamyl alcohol (PCI) volume, spinning rate (rpm), and precipitation agent on
quantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and swiftly transferred to a laboratory for DNA extraction. The lysis buffer used had
composition 5 M NaCl, 1 M Tris (pH=8.0), 0.5 M EDTA and20% SDS. The effect of various levels for each factor concerned was examined using General Linear Models or t-test procedures of SAS® software. The volume removed from the top aqueous part following the first and the second PCI washings was included in the models as a continuous variable; the variables of interest were OD280, OD260, OD260/OD280 (as quality criterion), total extracted DNA, extraction efficiency (ìg DNA/ìl blood), assay scores for easiness of removing the top aqueous phase after the first (assay 1) and the second (assay 2) spinning. The optimum level of factors significant for DNA extraction from fresh avian blood was found to be lysis buffer : blood sample ratio of 31:36 (ìl : ìl), incubation time of 60-70 min at 58oC, two washings with PCI at 1.2:1.3 PCI : top aqueous phase (ìl : ìl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 2000 - 2500 rpm for 20 min, precipitation of DNA with 1.5 – 2.0 volume of
absolute ethanol.
quantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and swiftly transferred to a laboratory for DNA extraction. The lysis buffer used had
composition 5 M NaCl, 1 M Tris (pH=8.0), 0.5 M EDTA and20% SDS. The effect of various levels for each factor concerned was examined using General Linear Models or t-test procedures of SAS® software. The volume removed from the top aqueous part following the first and the second PCI washings was included in the models as a continuous variable; the variables of interest were OD280, OD260, OD260/OD280 (as quality criterion), total extracted DNA, extraction efficiency (ìg DNA/ìl blood), assay scores for easiness of removing the top aqueous phase after the first (assay 1) and the second (assay 2) spinning. The optimum level of factors significant for DNA extraction from fresh avian blood was found to be lysis buffer : blood sample ratio of 31:36 (ìl : ìl), incubation time of 60-70 min at 58oC, two washings with PCI at 1.2:1.3 PCI : top aqueous phase (ìl : ìl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 2000 - 2500 rpm for 20 min, precipitation of DNA with 1.5 – 2.0 volume of
absolute ethanol.
