Plasmid DNA containing the insert to be transcribed should be purified by a CsCl gradient, or with commercially available chromatographic methods (e.g., Wizard or Qiagen columns). Crude alkaline lysis miniprep DNA contains unacceptable levels of ribonuclease and should not be used for in vitro transcription. For ssRNA: Plasmid templates should be linearized by digesting with a restriction enzyme that will cut the plasmid at a site downstream of the insert to be transcribed. For dsRNA: For simultaneous transcription of both strands encoded by a DNA insert, the plasmid containing T7 promoters on both sides of the polylinker should be linearized in separate reactions. The linearized plasmids should then be pooled prior to transcription. The restriction enzyme used to linearize the plasmid should leave blunt ends or 5´ overhangs (linearization of template with an enzyme that produces a 3´ overhang will result in aberrant transcripts). The quantity of DNA to be digested depends on the scale of the subsequent transcription reaction (see protocols for in vitro transcription). As a rough guide, 30 µg of plasmid DNA should be digested per milliliter of transcription reaction. DNA should be digested in a minimum volume of 10 µl per µg of DNA, using the buffer and temperature recommended for each enzyme. Confirm digestion by agarose gel electrophoresis and estimate quantity of DNA by comparison of band intensity to a known quantity of linearized marker DNA. The presence of small amounts (< 5%) of undigested plasmid will have little effect on RNA yield. Linearized DNA can be used straight from the digestion reaction if desired, with only a slight reduction of yield (< 10%). However, digestion reactions must be heat-denatured at 65°C for 20 minutes prior to transcription. Template linearized DNA from restriction digestion reaction should not constitute more than 10% of the total transcription volume.