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In vitro Transcription

In vitro Transcription: In Vitro Transcription

AmpliCap™ SP6 High Yield Message Maker Kit DISCONTINUED

For co-transcriptional capping of IVT RNA with SP6 RNA polymerase and standard cap analog. A reaction produces 35 µg RNA from 1 µg DNA in 2 hours. Reaction products are 40% correctly capped, 40% incorrectly capped and 20% uncapped.

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Description

The AmpliCap™ SP6 High Yield Message Maker Kit produces capped RNA by in vitro transcription using SP6 RNA polymerase and the standard cap analog m7G[5']ppp[5']G. A 2 hour, 20 μl AmpliCap reaction yields up to 35 μg of m7G-capped RNA from 1 μg of the 1.4 kb standard SP6 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.

CELLSRIPT also offers the AmpliCap-Max™ T7 High Yield Message Maker Kit and the MessageMAX™ T7 ARCA-Capped Message Transcription Kit, both of which produce unsurpassed yields of capped in vitro transcripts (up to 60 μg of RNA in a 30 minute standard control reaction with an 80% capping efficiency). The AmpliCap-Max Kit uses the standard RNA Cap Analog m7G[5']ppp[5']G and the MessageMAX Kit uses the anti-reverse cap analog m27,3'-OG[5']ppp[5'].

 

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Protocols

Protocols for: AmpliCap™ SP6 High Yield Message Maker Kit

Kit contents: AmpliCap™ SP6 Enzyme Solution (with added RNase inhibitor), AmpliCap™ 10X Transcription Buffer, AmpliCap™ Cap/NTP PreMix 20 mM GTP, RNase-Free DNase I, DTT, RNase-Free Water, Control Template DNA (linearised)

AmpliCap™SP6 High Yield Message Maker Kits Protocol

Please note: all protocols are the responsibility of the product supplier

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References

 

Citations for AmpliCap™ SP6 High Yield Message Maker Kit

  1. Agis-Juarez, R. A., et al. (2009) Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells, J. Gen. Virol. 90 , 2893-2901.
  2. Rother, S. & Strasser, K. (2007) The RNA polymerase II CTD kinase Ctk1 functions in translation elongation, Genes & Dev. 21 , 1409-1421.
  3. Nishimura, T., et al. (2005) The Arabidopsis STV1 Protein, Responsible for Translation Reinitiation, Is Required for Auxin-Mediated Gynoecium Patterning, Plant Cell 17 , 2940-2953.
  4. Blaney, J. E. J., et al. (2004) Genetically modified, live attenuated Dengue virus type 3 vaccine candidates, Am J Trop Med Hyg 71 , 811-821.
  5. Chiba, Y., et al. (2003) S-adenosyl-L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine {gamma}-synthase gene in Arabidopsis, PNAS 100 , 10225-10230.
  6. Hanley, K. A., et al. (2002) Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes, J. Virol. 76 , 525-531.

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Notes

Notes

  1. Use the AmpliScribe SP6 High Yield Transcription Kit to produce highest yields of uncapped RNA.
  2. Because of the very high concentration of NTPs used in AmpliCap™ reactions, these kits are not recommended for preparing high specific activity radiolabeled RNA. To generate high specific activity RNA probes, use the RiboScribe™ RNA Probe Synthesis Kits.

 

 




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