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) 0.5µl	  Rnasin 0µl	  water 5µl	  plasmid(1µg) or PCR (100-500ng) 2µl	  T7 polymerase 25.5µl total place at 37°C for 1-3 hrs. 1x buffer: 40mM Tris pH 8 6mM MgCl2 2mM spermidine 10mM NaCl

This particular method is originally from Krainer. (Danielle I need a cite)