Rapidly Synthesize Large Amounts Of Capped RNA

Rapidly Synthesize Large Amounts of Capped RNA

• Optimized for highest yield of capped RNA

• 80% capping efficiency

• Complete with SUPERase•In™ RNase inhibitor, DNase I, and positive control

• Ideal for microinjection, in vitro or in vivo translation

The mMESSAGE mMACHINE® High Yield Capped RNA Transcription Kits use Ambion's patented high yield transcription technology. Each reaction synthesizes 10-50 times the yield of capped RNA obtained with conventional in vitro transcription reactions.

The reaction is simple. DNA template is mixed with the reaction components and incubated at 37°C for up to 2 hours. Figure 1 shows a time course experiment that plots yield of a 3.7 kb capped transcript over time. With this template, the mMESSAGE mMACHINE reaction is complete in only 30 minutes. In addition to synthesizing high yields of capped RNA transcripts (Figure 1), mMESSAGE mMACHINE reactions also produce full-length transcripts. Figure 2 shows an Agilent bioanalyzer 2100 electropherogram of the 3.7 kb transcript. The presence of a single, strong peak indicates the predominance of full length transcript. This is also evident in Figure 3, in which mMESSAGE mMACHINE reactions producing 1.7 kb, 3.7 kb and 9 kb transcripts were analyzed on a conventional denaturing agarose gel.

Figure 1. mMESSAGE mMACHINE Reaction Time Course. A 3.7 kb template was transcribed in 3 replicate mMESSAGE mMACHINE reactions as per protocol. After the indicated incubation times, each reaction was DNase treated as per protocol and a preliminary quantitatation was performed with RiboGreen® (Molecular Probes). Each reaction was then purified using the MEGAclear™ Kit (Ambion) and quantitated by UV spectrophotometry.

Figure 2. Electropherogram of a mMESSAGE mMACHINE Reaction. A 3.7 kb template was used in a 20 µl mMESSAGE mMACHINE reaction as per protocol and incubated for 30 min followed by analysis using the Agilent 2100 bioanalyzer.

Figure 3. Gel Analysis of mMESSAGE mMACHINE Reactions. Templates generating transcripts of sizes 1.7 kb, 3.7 kb and 9 kb were used in a mMESSAGE mMACHINE reaction as per protocol. One µg of each reaction was denatured using NorthernMax®-Gly Sample Loading Dye and analyzed on a 1% agarose gel with EtBr.

Cap analog is premixed with GTP at a ratio optimized for the best compromise between yield and proportion of capped transcripts (80%). The cap analog:GTP ratio can be adjusted with the GTP Solution provided in the kit to increase yields of long transcripts (>4 kb); in fact, RNA transcripts as long as 23 kb have been synthesized with mMESSAGE mMACHINE. The kit includes SUPERase • In RNase Inhibitor . SUPERase • In inhibits RNases A, B, C, 1 and T1 and offers the greatest available protection against RNases (see data). DNase I and a positive control DNA template are also included. The RNA synthesized from a mMESSAGE mMACHINE reaction is ideal for in vitro or in vivo translation studies.

Ambion also offers the mMESSAGE mMACHINE T7 Ultra Kit for the generation of RNA transcripts capped with Anti Reverse Cap Analog (ARCA). A base modification in this cap analog generates RNAs with ARCA incorporated only in the functional, translatable orientation, resulting in higher protein yields. The mMESSAGE mMACHINE T7 Ultra Kit includes reagents for both transcription and the addition of a poly(A) tail to capped transcripts.

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Ordering Information
Cat# Product Name Size 1340 mMESSAGE mMACHINE® SP6 Kit 25 rxns 1344 mMESSAGE mMACHINE® T7 Kit 25 rxns 1345 mMESSAGE mMACHINE® T7 ULTRA Kit 10 rxns 1348 mMESSAGE mMACHINE® T3 Kit 25 rxns