1. Pipet 1 ml sddH2O into 1.5 ml microcentrifuge tube.
2. Add 3 ml of whole blood and incubate at room temperature 30 minutes.
3. Microcentrifuge at 15,000 RPM for 3 minutes.
4. Using P1000, carefully remove 970 ml of supernatant and dispose. Do not disturb pellet.
5. Place bottle of 5% Chelex on Magnestir.
6. Add 170 ml of stirring 5% Chelex to sample (final volume of 200 ml).
7. Incubate at 60°C for 30 minutes.
8. Preheat water bath to boiling.
9. Place sample in boiling water bath for 8 minutes.
At this point, samples may be stored at -20°C for up to one week. It is strongly recommended that extracted DNA be used as soon as possible. Perform steps 10-12 to prepare DNA for PCR.
10. Remove samples from freezer and thaw.
11. Vortex fast for 10 seconds.
12. Microcentrifuge: 3 minutes, 14000 RPM.
13. Use 20 ml of DNA suspension for PCR reaction (instead of the normal 1 ml). Collect only from the top of the tube. Proceed to Section 3. PCR.
If using Chelexed DNA, you will need to adjust your PCR Master Mix by
reducing the volume of water by 19 ml for each sample.