mMESSAGE mMACHINE™ Kit is designed for in vitro synthesis of large amounts of capped RNA. Capped RNA mimicsmost eukaryotic mRNAs found in the body because it has a 7-methylguanosine cap structure at the 5' end. The mMESSAGE mMACHINE™ Kit reaction includes the 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 prevents its incorporation at other sites in the RNA molecule.

How the mMESSAGE mMACHINE™ Kit Works
The mMESSAGE mMACHINE™ Kit has a simplified reaction format in which all 4 ribonucleotides and cap analogues are mixed in a single solution middle. The solution has a cap analog:GTP ratio of 4:1, which is suitable for maximizing RNA yield and capped transcript ratio. The mMESSAGE mMACHINE™ Kit is suitable for routine synthesis of capped RNA for oocyte microinjection,in vitro translation, transfection and other applications. High yields are achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide concentrations. Additionally, the synthesized RNA will not be degraded by any contaminating ribonucleases that may contain RNase inhibitors as part of the enzyme mix.

Using the mMESSAGE mMACHINE™ Kit
In a 20 µL reaction, high-yield capped RNA transcription from mMESSAGE mMACHINE™ will generate a large amount of capped RNA during a 2 hour incubation period. RNA. This method achieves yields 10–50 times higher than using conventionalin vitro transcription reactions. The ratio of cap analog to GTP has been optimized to provide a good compromise between yield (15–35 µg) and proportion of capped transcripts (80%). The mMESSAGE mMACHINE™ Kit also contains a LiCl precipitation solution, which efficiently separates protein from capped RNA and unincorporated nucleotides (including free cap analogs), improving translation efficiency. The mMESSAGE mMACHINE™ Kit is optimized for use only with the polymerase included in the kit. Using a different polymerase may result in poor yields. The mMESSAGE mMACHINE™ Kit contains all buffers and reagents required for 25 transcription reactions. Using the control template provided with the kit (Xenopus laevis elongation factor 1α, pTRI –25 µg RNA.