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內(nèi)容及儲(chǔ)存
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T7 Enzyme Mix, 10X Reaction Buffer 2XNTP?ARCA Solution, GTP Solution, pTRI-Xef, TURBO DNase, Ammonium Acetate Stop Solution, Lithium Chloride Precipitation Solution,?E-PAP, 5X?E-PAP Buffer, ATP Solution, MnCl2, and Formaldehyde Load Dye are all stored at –20°C. Nuclease-free Water may be stored at any temperature.
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描述
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The mMESSAGE mMACHINE? T7 Ultra Kit combines a new cap analog, anti-reverse cap analog (ARCA), with a patented high-yield transcription technology to generate RNA transcripts that produce higher protein yields compared to other transcripts upon translation. Advantages of the mMESSAGE mMACHINE? T7 Ultra Kit:? Synthesize more protein from?in vitro-transcribed RNA? Express RNA transcripts more efficiently both?in vitro?and?in vivo? Stabilize transcripts?in vivo?with included reagents for poly(A) tailingARCA-capped RNA is translationally more activeA base modification in ARCA results in its incorporation in the functional, translatable orientation only; traditional cap analog can be incorporated in both functional and nonfunctional orientations. As a result, incorporating ARCA into transcription reactions yields capped RNAs that are 100% translatable. This is further enhanced by the inclusion of poly(A) tails in mMESSAGE mMACHINE? T7 Ultra transcripts. Experiments comparing ARCA and ARCA/poly(A)-tailed transcripts to cap analog and cap analog/poly(A)-tailed transcripts indicate higher levels of protein synthesis with ARCA capped RNA .What Is ARCA?ARCA is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with OCH3?(see schematic). Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group, thus forcing ARCA incorporation in the forward orientation. As a result, 100% of the transcripts synthesized with ARCA at the 5' end are translatable, leading to a strong stimulatory effect on translation.Proper capping of?in vitro?transcribed RNAProper capping of RNA promotes correct initiation of protein synthesis, as well as stability and processing of mRNA?in vivo. Uncapped RNA is rapidly degraded by cellular RNases after microinjection or transfection into cells. Capped RNA is also typically translated more efficiently in?in vitro?translation systems, generating RNAs with cap analog incorporated only in the functional orientation. Therefore, substitution of traditional cap analog with ARCA results in the synthesis of capped RNAs that are 100% translatable.