The linear control DNA supplied with the system produces single-stranded transcripts that are 1.1kb and 2.3kb in length. The transcripts produced from the pGEM? Express Positive Control Template are not complementary and thus will not form double-stranded RNA. They are to be used as a positive control for transcriptional activity of the system and RNA integrity following transcription.
1. Set up the appropriate reaction size at room temperature. The 20μl reaction may be scaled as necessary (up to 500μl total volume; use multiple tubes for reaction volumes >500μl). Add the components in the order shown.
T7 Reaction Components
|RiboMAXTM Express T7 2X Buffer*
|liner Template DNA(`1μg total)
|pGEM® Express Positive Control Template
|Enzyme Mix,T7 Express
*Frozen RiboMAX™ Express T7 2X Buffer will contain a precipitate that can be dissolved by warming the buffer at 37°C and mixing well. Note that the buffer contains spermidine, which can precipitate DNA at temperatures colder than room temperature.
2. Mix gently and incubate at 37°C for 30 minutes (see Notes 1–3).
1) For plasmid templates, up to 3μg of purified template may be included per 20μl reaction.
2) To maximize yield, incubation at 37°C may proceed for up to 2–6 hours. In general, however, no dramatic increase in yield is observed beyond the 30-minute incubation period, except for smaller transcripts (~200 bases). A time-course experiment may be performed to determine the optimal
incubation time for maximal RNA synthesis.
3) Incubation at 42°C may improve the yield of dsRNA for transcripts that contain secondary structure. If sufficient yield is not obtained at 37°C and the template is GC-rich, incubate the reaction at 42°C instead.