Synthesis of small RNAs using T7 RNA polymerase

This chapter discusses how transcription templates are prepared by cleaving the plasmid DNA with a restriction enzyme of choice. The individual nucleoside triphosphates (NTPs) are diluted to desired concentrations and are mixed in equimolar ratios to make a stock solution. If they are supplied in ethanol and will be used in large amounts, the ethanol should be removed prior to use. The addition of serum albumin to the reaction is optional and is generally omitted in large-scale reactions. Differences in template sequence and length can result in substantial differences in the optimal enzyme and template concentrations required for the production of RNA. In addition, one or more of the components may be more valuable than the others. It is, therefore, worthwhile to carry out trial reactions to find optimal reaction conditions for a given synthesis. Although performing trial reactions to optimize reaction conditions can be tedious, reaction yields can be substantially higher than those obtained using the general conditions and the reactions scale up readily.