FAQ: What are the main causes of reaction failure using SP6 RNA Polymerase?

*Dithiothreitol is required for activity. Buffer older than 6 months should be supplemented with fresh DTT.
*SP6 is extremely sensitive to salt inhibition. Use drop dialysis or other purification to remove salt from DNA preparations.
*RNase contamination degraded the RNA product. Use RNase inhibitor at 1 unit/ml (provided in the HiScribe T7 In Vitro  Transcription Kit (NEB# E2030 ). 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.