FAQ: What are the main causes of reaction failure using T7 RNA Polymerase?

1. Dithiothreitol is required for activity. Buffer older than 6 months should be supplemented with fresh DTT.
2. T7 RNA Polymerase is extremely sensitive to salt inhibition. Use drop dialysis or other purification to remove salt from DNA preparations.
3. RNase contamination degraded the RNA product. Use RNase inhibitor at 1 unit/ml (provided in the HiScribe RNAi Transcription Kit (NEB# E2000)). Use ultra pure water and make sure the DNA template has been phenol/chloroform extracted. Do not use a gel loading buffer containing SDS because it inactivates the RNase inhibitor.