| 1. | Estimate the number of organisms/ml from your culture (typically ~ 108/ml). |
| 2. | Prepare three (3) dilutions of the culture with isotonic saline so that the concentrations are 103, 104, 105 organisms/ml. |
| 3. | Place 20ul of resuspended GeneReleaser® in each of three standard 1.5ml centrifuge tubes. |
| 4. | Add 1ul of each dilution of the yeast cultures prepared in #2 above to one of the GeneReleaser® containing tubes. Label each tube. |
| 5. | Using the disposable pestle provided, grind the yeast cells in the presence of GeneReleaser® using 10 turns and thrusts of the pestle. Drain the pestle into the tube. |
| 6. | Discard the pestle. |
| 7. | Vortex the tubes and transfer the entire contents to a fresh 0.5ml amplification tube. |
| 8. | Overlay each tube with 35-50ul of mineral oil. Close the tubes. |
| 9. | Place tubes in a microwave safe rack. |
| 10. | Heat the tubes at maximum power for 7-10 minutes. |
| 11. | Transfer the tubes to a preheated thermocycler at 80°C for 5 minutes. |
| 12. | Add 80ul of 1.25 X master mix and begin amplification sequence immediately after adding master mix to the last tube. |
| 13. | It is very important that the first denaturation cycle be for 4 minutes at 94°C. |
| 14. | Following amplification analyze the products per standard protocols. |
| 15. | Evaluate the amplification results and for future amplifications employ the dilution of yeast cells giving the best results. |
| 16. | The preceding protocol is for 100ul reactions. Both GeneReleaser® and master mix volumes can be proportionately reduced for smaller reaction volume, i.e. 50ul and 25ul amplificatons. |
