Part of #Optimizing factors influencing DNA extraction from fresh whole avian blood# :
Publishing year : 2005
Conference : Fourth National Biotechnology Congress of Iran
Number of pages : 4
Abstract: A study was conducted to optimize the effective combination of lysis buffer, Proteinase K, incubation time, phenol-chloroform isoamyl alcohol (PCI) volume, spinning speed (rpm), and precipitation agent on the quantity and quality of DNA extracted from the whole form avian blood Whole blood samples were collected in EDTA and quickly transformed into a laboratory for DNA extraction. The lysis buffer used had a composition of 5M NaCl, 1M Tris (pH = 8.0), 0.5M EDTA, and 20% SDS. The effect of different levels for each of the above-mentioned factors has been investigated using SAS software's General Linear Models or t-test procedures. The volume that was removed from the top aqueous part after the first and second PCI washings was included in the models as a co-variant. The variables of interest were composed of OD280, OD260, OD260 / OD280 (as quality criterion), total extracted DNA, extraction efficiency (g DNA / l blood), assay scores for the ease of removing the top aqueous phase after first (assay 1) and second (assay 2) spinning. The optimum level of each factor for DNA extraction from whole fresh avian blood was found to be lysis buffer: blood sample ratio = 31-36 (l: l), incubation time = 60-70 min at 59-60 C, two times washing with PCI at 1.2-1.3 PCI: top aqueous phase (1: 1) for the first and 1.4 for the second washing, centrifugation of the homogenized sample at 10,000 rpm for 10-20 min, precipitation of DNA with 1.5-2.0 volume absolute ethanol.