For short templates we recommend to increase in template concentration, polymerase concentration and reaction time, since transcription of small RNAs is largely dependent on the number of initiation events.
RNase-Free Water: µl
10X Transcription Buffer: 4 µl
20X Ribonucleotide Solution Mix: 2 µl
Template (0.3–0.5 µg): µl
20X HMW Mix: 2 µl
T7 RNA Polymerase (500 units/µl): 4 µl
Total reaction volume: 40 µl
Incubate at 42°C for at least 3–4 hrs. Overnight incubations will result in higher yields.
Transcription yields increase linearly for the first 90 minutes (approximately) and reach maximum after 2–3 hours. Reactions can be carried out overnight if desired, but yields will not be higher (except when transcribing RNA shorter than 0.5 kb). Double-stranded RNA is stable upon prolonged incubation at 42°C.
The volume of template used in the transcription reaction depends on the method of purification. For unpurified, heat-killed restriction digests, include no more than 13 µl of template per 40 µl reaction. For unpurified PCR product, include no more than 7 µl per 40 µl reaction. In all cases, the amount of added template DNA should not exceed 2 µg per 40 µl reaction, as RNA yields will not be higher at template concentrations greater than this.
While all of the components in the HiScribe T7 In Vitro Transcription Kit are RNase-free, it is possible to introduce ribonucleases into the transcription reaction from the laboratory environment. This can be avoided by following some simple precautions: 1) always wear gloves when working with RNA, 2) use either a dedicated set of pipettors for RNA work or aerosol-resistant (barrier) pipette tips, 3) use ultrapure water (Milli-Q or equivalent) and autoclave all solutions if possible, and 4) use disposable plasticware instead of glassware whenever possible. It should NOT be necessary to treat solutions and equipment with diethyl pyrocarbonate (DEPC). DEPC can inhibit subsequent reactions if it is not completely inactivated following treatment.