Puregene

DNA Isolation for whole blood

Experiment is divided into 4 stages.

Aprpecitaoin for this information is over 9000-thank you!

[edit] Stage 1 - Cell Lysis

Add 1ml whole blood to a 15ml tube containing 3 ml of RBC Lysis Solution. Invert to mix and incubate at room temperature for 10 mins. Invert again at least once during the incubation.
Centrifuge at 2000 x g for 10m. Discard supernatant leaving behind the white cell pellet and about 100-200 ul of the residual liquid.
Vortex the tube vigorously to suspend the cells in the residual liquid. This greatly facilitates cell lysis in step #4 below.
Add 1 ml of Cell Lysis Solution to the resuspended cells and pipette up and down to lyse the cells. Usually no incubation is required, however, if cell clumps are visible after mixing, incubate at 37^oC or room temperature until the solution is homogenous. Samples are stable in Cell Lysis Solution for at least 18 months at room temperature.

Cool the sample to room temperature.
Add 333ul of PPS to the cell lysate.
Vortex vigorously at high speeds for 20 secs to mix the PPS uniformly with the cell lysate.
Centrifuge at 2000 x g for 10m. The precipitated proteins will form a tight dark brown pellet. If the protein pellet is not tight, repeat step 3 followed by incubation of ice for 5m and then repeat step #4.

Transfer the supernatant containing the DNA into a 15ul tube containing 1ml of 100% Isopropanol.
Mix the sample by inverting gently for about 50 times.
Centrifuge at 2000 x g for 3m; the DNA will be visible as a small white pellet.
Discard supernatant and drain tube briefly on clean absorbent paper. Add 1ml of 70% Ethanol and invert tube several times to wash the DNA.
Centrifuge at 2000 x g for 1m. Carefully use a pipette to remove the DNA (white pellet) from the ethanol, and transfer into another clean tube.
Air dry the tube containing DNA in a speed-vac for about 10m.