Berglund:Hot, Capped RNA Method 1
Transcription used to make RNAs for splicing. The M7cap is a GTP capping nucleotide that helps prevent degregation of the RNA during splicing. Some papers have cited using an unmethylated cap, I found it works just as well as a methylated cap. I have also tried not capping the RNA and I have noticed capped being better than uncapped. The CTP is from Perkin-Elmer/NEN. The NTP's need to be pHed to 8, and the reaction needs to be a final pH8.1 at 37°C (pH changes with temperature changes). pH and T7 buffer are most often the reason why this reaction fails (other than template problems). The spermidine in the buffer does not last a long time and as it breaks down your amt of product decreases. Milligan and Uhlenbeck, 1989, Methods in Enzymology: 180 51-62 is an excellent source to be read by the serious transcriber.
5µl 5xT7 buffer
2µl 400mM DTT
2.5µl rNTP Mix (5mM A, U, 1mM G)
2.5µl 0.1mM CTP
5µl P32 CTP (BLU-508H)
1µl 10mM M7cap (Ambion: 8050)
5µl plasmid(1µg) or PCR (100-500ng)
2µl T7 polymerase
place at 37°C for 1-3 hrs.
40mM Tris pH 8
This particular method is originally from Krainer. (Danielle I need a cite)