| 1. | Take an aliquot of cells (1,000,000) and transfer to PCR tube. The cell # should be such that 1-10µl will contain the 106 cells. |
| 2. | Add 20µl of GR and vortex. |
| 3. | Overlay with RNase free mineral oil. |
| 4. | Follow microwave protocol. |
| 5. | Add sufficient RNase free DNase to digest DNA if DNA will interfere with your PCR or RT-PCR. We use 0.1 to 1 unit of DNase per tube and incubate 1 hour at 37°C. |
| 6. | Destroy DNase by addition of 0.1 units of Proteinase K and heating at 55°C for 30 minutes. Alternatively, DNase can be heat inactivated by 94°C for 10 minutes. |
| 7. | Destroy Proteinase K activity by heating to 94°C for 10 minutes. |
| 8. | Centrifuge tubes for 2 minutes at 10,000xg to form a tight GR pellet. |
| 9. | Perform RT directly in tube limiting reagent volumes to no more than 50µl total volume. Follow manufacturer's protocol for RT. |
| 10. | Perform PCR in the tube containing the cDNA produced by RT. We recommend a 100µl PCR. Note: Adjust buffers, Mg, etc., so that the final PCR volume is 100µl and all components are 1X and RNase free. |
