T7体外转录试剂盒 加帽mMESSAGEmMACHINE T7 kit (Ambion(5)

Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

IMPORTANT!Before using this product, read and understand the information in the “Safety” appendix in this document.

Introduction

BackgroundmMESSAGE mMACHINE® Kits are designed for the in vitro synthesis of large

amounts of capped RNA. Capped RNA mimics most eukaryotic mRNAs found in vivo, because it has a 7-methyl guanosine cap structure at the 5'end. mMESSAGE mMACHINE® Kit reactions include cap analog [m7G(5')ppp(5')G] in an ultra high-yield transcription reaction. The cap analog is incorporated only as the first or 5' terminal G of the transcript because its structure precludes its incorporation at any other position in the RNA molecule. mMESSAGE mMACHINE® Kits have a

simplified reaction format in which all four ribonucleotides and cap analog are mixed in a single solution. The cap analog:GTP ratio of this solution is 4:1, which is optimal for maximizing both RNA yield and the proportion of capped transcripts. mMESSAGE mMACHINE® Kits are ideal for the routine synthesis of capped RNAs for oocyte microinjection, in vitro translation, transfection and other applications.

The high yields are achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide concentrations. In addition, the RNA synthesized is protected from degradation by any contaminating ribonucleases that may be present with RNase inhibitor—a component of the Enzyme Mix. The mMESSAGE

mMACHINE® Kit contains all the buffers and reagents necessary for 25transcription reactions. Using the control template supplied with the kit (Xenopus elongation factor 1α, pTRI Xef), each mMESSAGE mMACHINE® reaction will yield approximately 20–30µg of RNA using T3 or T7 RNA polymerase, or about 15–25µg RNA using SP6 RNA polymerase.

mMESSAGE mMACHINE® Kit User Guide5