This protocol is for in vitro transcription of long RNAs off plasmid or PCR product templates. It is assumed that the template has been gel purified, is the right size, has a T7 promoter and a 3' polyA tail (typically A60). Following this protocol the RNA will be ready for transfection into mammalian cells or in vitro translation. Significant amounts of template are necessary: at least 1ug of template per 100ul reaction; ideally closer to 5ug of template, especially for very long templates. Use RNase sensitive protocols and reagents for all steps of this procedure. The basic protocol is: - PCR amplification of the template- in vitro transcription- capping and 2'-O-Methylation - quality control and optional purification Based on protocols from Kaihong Zhou (Doudna lab) and RNA: A Laboratory Manual (Rio, Ares, Hannon, Nilsen).