1: In Vitro Transcription of Long Templates (> 0.3 kb) (E2030)
This protocol may be used for any large-scale transcription reaction. Yields of ~1.5 mg per ml of transcription reaction should be easily obtainable using the HiScribe T7 In Vitro Transcription Kit. A 40 µl pilot reaction should be carried out initially to test the quality of DNA template and transcription reagents. Reactions can then be scaled up accordingly as required by the particular application. Pilot reactions should be carried out side-by-side with reactions containing the StuI Linearized Litmus 28iMal Control Plasmid.
Protocol1. Transcription Reaction
- Thaw the 10X transcription buffer and 20X NTP mix at room temperature for the minimum amount of time required for complete thawing. Do not thaw at 37°C. If precipitant is evident following thawing, vortex briefly to resuspend. Keep the 20X High Molecular Weight Component (HMW) Mix and T7 RNA Polymerase at -20°C until needed.
- Combine the following, in order, taking caution to avoid ribonuclease contamination. Set up separate reactions for each plasmid of interest, as well as the StuI Linearized Litmus 28iMAL Control Plasmid.
RNase-Free Water: µl
10X Transcription Buffer: 4 µl
20X Ribonucleotide Solution Mix: 2 µl
Template(s) (1–2 µg): µl
20X HMW Mix: 2 µl
T7 RNA Polymerase (500 units/µl): 2 µl
Total reaction volume: 40 µl
- Incubate at 42°C for 2–4 hrs.
- For analysis of transcription run 1 µl of transcription reaction in a 1% agarose gel. The expected length of the transcript from the control template (StuI Linearized Litmus 28iMal Control Plasmid) is 901 nt.
- If the yield of RNA from the pilot reaction is sufficient, scale-up the reaction appropriately. Use no more than 500 µl per tube; use multiple tubes if necessary.
- The RNA product can be purified either by phenol/chloroform extraction and ethanol precipitation, isopropanol precipitation or by using a commercial kit for RNA purification.