This method provides a high-quality preparation of high molecular weight genomic DNA from whole blood. Proteinase K is used to liberate nucleic acids from cells.
10 ml collected EDTA blood is mixed with 30 ml DNA lysis buffer and left in a 50 ml polypropylene screw-cap tube (Falcon) on crashed ice for one hour. After centrifuging for 15 minutes at 1500 rpm (4℃C), the supernatant is decanted, the pellet (white blood cells) resuspended in 10 ml DNA lysis buffer and centrifuged again for 10 minutes at 1500 rpm (4℃C). The supernatant is decanted again and the pellet resuspended in 5 ml 1Õ SE buffer. 0.25 mg Proteinase K and 250 µl 20% SDS are added and incubated overnight at 37℃C.
1.5 ml saturated NaCl (6M) are added, shaken and centrifuged for 20 minutes at 17000 rpm. The supernatant is decanted into a fresh tube, centrifuged once again and transferred to a fresh tube again. Two volumes absolute alcohol are added to precipitate DNA2. Large pieces of DNA can be removed by collecting fibres with a glass rod and transferring them to a new tube. Again, 1 ml absolute ethanol is added, centrifuged and the supernatant decanted. The pellet is washed with 200µl 70% ethanol, centrifuged shortly, decanted and desiccated in a vacuum evaporator (Speed Vac).
Resampled DNA for the second study (Chapter 4) was prepared by using the Nucleon DNA Isolation Kit for mammalian blood (Amersham, England), following the supplied protocol. The kit procedure starts with preferential lysis of erythrocytes. The remaining leukocytes are lysed with a strong anionic detergent. Following cell lysis, deproteinisation with sodium perchlorate and a single partition against chloroform, the Nucleon proprietary resin is added. This resin binds proteins by the formation of an imide bond and, additionally, forms a semi-solid stratum during partitioning, which traps proteinaceous material at the interface and in the organic phase. This facilitates removal of the aqueous phase, ensuring excellent recovery of high quality DNA.
© 2001 Alexander Binder