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mMESSAGE 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. How mMESSAGE mMACHINE® kits work 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. Using mMESSAGE mMACHINE® Kits In a 20 µL reaction during a 2 hour incubation, mMESSAGE mMACHINE® High Yield Capped RNA Transcription will yield large mass amounts of capped RNA. Up to 10–50 times the yield obtained with conventional in vitro transcription reactions. The ratio of cap analog to GTP has been optimized to allow the best compromise between yield (15–35 µg) and proportion of transcripts that are capped (80%). mMESSAGE mMACHINE® Kits also contain a LiCl precipitation solution that is efficient for separating proteins and unincorporated nucleotides (including free cap analog) from the capped RNA, allowing an increased efficiency of translation. mMESSAGE mMACHINE® kits are only optimized for use with the polymerases included in the kit. Using a different polymerase may result in low yields. The mMESSAGE mMACHINE® kits contain all the buffers and reagents necessary for 25 transcription reactions. Using the control template supplied with the kits (Xenopus elongation factor 1α, pTRI Xef), each mMESSAGE mMACHINE® reaction will yield approximately 20–30 µg of RNA using T3 or T7 RNA polymerase, or about 15–25 µg RNA using SP6 RNA polymerase. |
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