The AmpliCap-Max™ T7 High Yield Message Maker Kit produces capped RNA by in vitro transcription using T7 RNA polymerase and the standard cap analog m7G[5']ppp[5']G. A 30 minute, 20 μl AmpliCap-Max reaction yields up to 60 μg of m7G-capped RNA from 1 μg of the 1.4 kb standard T7 control template DNA.

The m7G[5']ppp[5']G cap analog/NTP PreMix contains all four ribonucleotides and the monomethylated cap analog. The PreMix ensures the optimal concentration of each NTP and ratio of methylated cap analog to GTP (4:1), maximizing transcript capping (~80%) and yield. Because the concentration of GTP in the reaction is limiting, the cap analog is preferentially incorporated as the first or 5'-terminal G of the transcript.

CELLSCRIPT also offers the MessageMAX™ T7 ARCA-Capped Message Transcription Kit which also produces up to 60 μg of capped RNA in a 30 minute standard control reaction with an 80% capping efficiency. The MessageMAX Kit uses the anti-reverse cap analog (ARCA), m27,3'-OG[5']ppp[5'].

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Protocols for: AmpliCap-Max™ T7 High Yield Message Maker Kits

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• Igarashi, H., et al. (2010) 2A protease is not a prerequisite for poliovirus replication, J. Virol. , JVI.02575-09.
• Mahias, K., et al. (2010) Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis, Nucleic Acids Res. , gkq109.
• Nicholson, B. L., et al. (2010) Tombusvirus recruitment of host translational machinery via the 3' UTR, RNA 16 , 1402-1419.
• Sergeeva, O. A., et al. (2010) Fragrant Dioxane Derivatives Identify {beta}1-Subunit-containing GABAA Receptors, J. Biol. Chem. 285 , 23985-23993.
• Levy, S., et al. (2009) Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels, J. Biol. Chem. 284 , 32434-32443.
• Inoue, K., et al. (2008) Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model, Am J Physiol Gastrointest Liver Physiol 294 , G660-668.
• Gerbasi, V. R., et al. (2004) Yeast Asc1p and Mammalian RACK1 Are Functionally Orthologous Core 40S Ribosomal Proteins That Repress Gene Expression, Mol. Cell. Biol. 24 , 8276-8287.

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• If the capped RNA is to be used for translation studies, translational efficiencies can be much higher if the mRNA is also polyadenylated, especially if the mRNA is intended for use in vivo following transfection or microinjection. RNA can be easily and efficiently polyadenylated using EPICENTRE's Poly(A) Polymerase Tailing Kit.
• Use the AmpliScribe™ T7-Flash™ and the AmpliScribe SP6 High Yield Transcription Kit to produce the highest yields of uncapped RNA.

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Applications

• Generate 5´-capped RNA with a standard cap, from DNA cloned downstream of a T7 or T3 RNA Polymerase promoter, for in vitro translation and microinjection.
• Functional studies of heterogenous nuclear RNAs and viral RNAs.

Benefits

• Yield up to 60 µg of RNA from 1 µg of control templates in a 20-µl reaction in 30 minutes.
• Capping efficiencies up to 80%.
• An optimized m7G[5´]ppp[5´]G Cap/NTP PreMix is provided for ease of use and highest yields of capped RNA transcripts.
• A separate vial of GTP is provided for efficient production of long, 5´-capped RNA.
• Formulated to use high concentrations of NTPs that are inhibitory to conventional in vitro transcription kits.
• RNase inhibitor included.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200