| 1. | Obtain plasma or serum by standard collection techniques. |
| 2. | For maximum sensitivity, treat 5µl of serum with 15µl of resuspended GeneReleaser®. |
| 3. | Vortex the mixture briefly, i.e. 1-2 seconds. |
| 4. | Overlay with mineral oil. |
| 5. | Treat the specimens using either the thermocycle lysis program or by the microwave protocol. |
| 6. | For best results, after conducting the lysis program in 5 above, place the tubes containing the treated specimens on the thermocycler and heat to 80°C. |
| 7. | For a 100µl final volume amplification add 80µl of 1.25X Master Mix containing all components for the amplification except the target source DNA. For smaller amplification adjust the volume and concentration of the amplification reagents proportionately. |
| 8. | Amplify the specimens according to your optimized protocol noting the following: |
| |
- The first cycle should have a denaturing time of 2-4 minutes at 94°C.
- The annealing temperature should be no higher than 55°C.
- For some primer sets a retitration of the magnesium concentration may be required.
- Appropriate controls treated with GeneReleaser® should also be analyzed.
- For very low copies of the virus 40-50 cycles of amplification may be required.
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| Note: If serum aggregation occurs (esp. with microwave protocol), we suggest the addition of fetal calf serum at a ratio of 1:1 with your serum. |
