| 1. | Collect whole blood by standard venipuncture or other techniques observing appropriate protection protocols. EDTA, Heparin and ACD preservatives are suitable. |
| 2. | Transfer an aliquot of whole blood with a population of 106 nucleated cells (approximately 200 ul) to a .5ml amplification tube. (NOTE: DO NOT USE ANOTHER SIZE TUBE!). |
| 3. | Add to the WB aliquot an equal volume of 0.14M NH4Cl, 0.017 M TRIS, pH 7.4. Vortex this mix 5 seconds. Place on ice or at 4°C for 5 minutes. |
| 4. | Remove tubes from ice. If variable speed microfuge is used, centrifuge at 1,000xg for 1 minute. If fixed speed microfuge is used, pulse in 3 second intervals for 12 seconds or 4X. |
| 5. | Remove and discard the supernatant containing the RBC lysate being careful not to dislodge the cell pellet. |
| 6. | Wash the remaining pellet 3 times with 200 ul of 1x PCR buffer (whatever type you will be amplifying with) with 1.5 mM MgCl2. Vortex briefly and microfuge as per steps 4 and 5 above and discard the supernatant. |
| 7. | To the remaining pellet, add 20ul 1xTE and 20ul GeneReleaser®. |
| 8. | Vortex mix of step 6 for 5 seconds, then microwave in a suitable rack for 7 minutes on "High" (4500 Watt-Minutes). |
| 9. | Centrifuge 30 seconds, draw off 20ul of the supernatant which now contains the DNA/RNA released from cells, and transfer this to a fresh tube and bring up to a total volume of ~200ul with the addition of 180ul of 1xTE. |
| 10. | Use 5ul of this DNA solution for each 100ul reaction. Use for PCR or store at 4°C for later PCR. Amplification can be successfully performed on specimens processed by this protocol and stored at 4°C for over 60 days. Data suggests that specimens processed as per above can be amplified after at least one year when stored at -20°C. |
