| 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. | Following the lysis treatment, add an additional 20µl of RNase free water to the tubes and centrifuge at 12,000 x g for 5 minutes. |
| 7. | Transfer 10-20µl of the supernatant from step 6 to a fresh tube. |
| 8. | Perform reverse transcriptase reaction according to the source of your reverse transcriptase enzyme vendor's protocol. |
| 9. | Using the cDNA obtained in step 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 60°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.
|
| 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. |
