The AmpliScribe™ T7 High Yield Transcription Kit is specially formulated to use high concentrations of NTPs, that can be inhibitory to other in vitro transcription systems. AmpliScribe High Yield Transcription reactions can produce >20-fold more full-length RNA transcript than conventional in vitro transcription reactions. For example, the AmpliScribe T7 Kit yields approximately 5-7.5 mg/ml of 1.4-kb full-length control RNA in 30-120 minutes, respectively, using 1 µg of template DNA (Figure. 1).

Applications

• High-yield synthesis of RNA from DNA cloned downstream from a T7 RNA polymerase promoter, for microinjection, RNA interference, or antisense experiments.
• Structural and functional studies of splicing, processing, heterogeneous nuclear RNAs, tRNAs, viral RNAs, and ribozymes. ¹
• Production of fluorescent ²-, biotinylated ³-, or digoxigenin ⁴- labelled RNA.

Features

• High yield. Produce microgram amounts of RNA from all templates—synthesise both long (Figure. 2) and short RNA using only one kit. ⁵
• Sensitive. Produce microgram amounts of RNA from as little as 1 ng of template.
• Scalable. Increase the reaction size to produce milligram amounts of RNA.
• Flexible. Substitute a labelled nucleotide for one of the supplied NTP solutions.
• Convenient. RNase inhibitor included.

Note: Because of the very high concentration of NTPs used in AmpliScribe reactions, these kits are not recommended for preparing high specific-activity radiolabelled RNA probes.

Figure 1. The AmpliScribe™ T7 High Yield Transcription Kit is specially formulated to consistently produce >20-fold more RNA than a conventional T7 in vitro transcription reaction.

Figure 2. An AmpliScribe™ T7 High Yield Transcription reaction produces full-length, long RNA transcripts. Aliquots from standard 20 µl AmpliScribe T7 High Yield Transcription reactions were loaded onto a denaturing 1% agarose-formaldehyde gel ⁶ and stained with ethidium bromide. The size of the RNA transcripts and RNA yields (in parentheses) were: Lane 1, 3.0 kb (185 µg); lane 2, 4.0 kb (178 µg); lane 3, 5.0 kb (176 µg); lane 4, 6.0 kb (169 µg); lane M, RNA size marker.

References

1. Johnson, M. (1995) Epicentre Forum 2(5), 4.
2. DeLong, E.F. et al. (1999) App. And Environ. Microbiol. 65, 5554.
3. Kaplan, E. et al. (1996) Epicentre Forum 3(2), 1.
4. Hoff man, L.M. and Johnson, M.G. (1994) BioTechniques 17, 372.
5. Schanke, J. (2000) Epicentre Forum 7(2), 6.
6. Molecular Cloning - A Laboratory Manual, Third Edition, 2001. CSHL Press. pp 7.27 - 7.34. J. Sambrook and D. Russell.