Berglund:Hot, Capped RNA Method 2

Transcription used to make RNAs for splicing. This method produces more RNA that is not as hot as Method 1. This has usefulness at certain times. The capping ratio is higher to make sure the RNA gets capped. 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. 4µl 5X T7 buffer 2.5µl template 4µl water 2µl 400mM DTT 1µl NTP's 1.5µl p32 CTP 1µl 40mM m7GpppG 5' GTP cap 0.5µl 1M Tris pH 9 1µl 10xyeast pyrophosphatase 0.5µl RNASIN 1.5µl T7 polymerase(25U) 1x buffer: 40mM Tris pH 8 6mM MgCl2 2mM spermidine 10mM NaCl fc= 400µM ATP fc= 400µM UTP fc= 75µM CTP fc= 100µM GTP fc=2000µM GTP cap fc=75µCuries CTP