The HiScribe T7 High Yield RNA Synthesis Kit is an extremely flexible system for in vitro transcription of RNA using T7 RNA Polymerase. The kit allows for synthesis many kinds of RNA including internally labeled and co-transcriptionally capped transcripts.RNA synthesized from the kit is suitable for many applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis, microinjection, and in vitro translation and RNA vaccines.The kit contains sufficient reagents for 50 reactions of 20 μl each. Each standard reaction yields up to 180 μg of RNA from 1 μg control template. Each kit can yield up to 9 mg RNA. For 32P labeling, the kit contains enough reagents for 100reactions of 20 μl each.Materials Not Included:
DNA Template: The DNA template must be linear and contain the T7RNA Polymerase promoter with correct orientation in relation to target sequence to be transcribed.
3′-O-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1411)
m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1404)
m7G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1405)
G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1406)
G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1407)
Modified-NTP: Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP
General: 37°C incubator or PCR machine, nuclease-free water
DNase I: DNase I (RNase-free) (NEB #M0303)
Purification: Buffer- or water-saturated phenol/chloroform, ethanol and 3 M sodium acetate, pH 5.2, spin columns
Gel Analysis: Gels and running buffers, gel apparatus, power supply
Figure 1. Transcription by T7 RNA PolymeraseFigure 2. Time course of standard RNA synthesis from three DNA templatesReactions were incubated at 37°C in a PCR machine. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.Figure 3. Effect of template amount on RNA yieldStandard reactions were incubated at 37°Cin a PCR machine for 2 hours. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.Figure 4: Improved RNA yield and integrity from extended duration transcription reactionsreactions were assembled, in duplicate, according to the manufacturers’ suggested protocols using 3 ng of dsDNA template encoding a 1.8 kb RNA, and incubated at 37°C for 16, 24 and 40 hours. At each time point, the corresponding tubes were transferred to -20°C to stop the reaction. Transcription reactions were column purified after the last time point.(A) Transcript yield – After column purification, RNA concentration was measured using a NanoDrop spectrophotometer and total RNA yield was calculated. These data demonstrate that a substantially higher yield of RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.(B) Transcript integrity – 150 ng of column purified RNA was run a 1.2% denaturing agarose gel, stained with ethidium bromide and visualized by UV fluorescence. The data demonstrate greatly improved transcript integrity after extended duration RNA synthesis reactions using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.
This product is related to the following categories:
RNA Synthesis Products
This product can be used in the following applications:
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