New England Biolabs Canada

HiScribe® T7 High Yield RNA Synthesis Kit

Catalog # Concentration Size
E2040L 250 reactions
E2040S 50 reactions
Catalog # Concentration Size
E2040L 250 reactions
E2040S 50 reactions
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    Avoiding RNase Contamination
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 100 reactions of 20 μl each. 

Materials Not Included:
  • DNA Template: The DNA template must be linear and contain the T7 RNA 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 
  • Labeling: [α-32P] labeled ribonucleotide (800-6,000 Ci/mmol) 
  • 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 Polymerase Figure 1. Transcription by T7 RNA Polymerase



Figure 2. Time course of standard RNA synthesis from three DNA templates Figure 2. Time course of standard RNA synthesis from three DNA templates

Reactions 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 yield Figure 3. Effect of template amount on RNA yield

Standard 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 reactions Figure 4: Improved RNA yield and integrity from extended duration transcription reactions

Reactions 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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  CTP  N0454AVIAL -20 1 x 0.1 ml 100 mM
  FLuc Control Template N0426AVIAL -20 1 x 0.01 ml 0.5 µg/µl
  UTP  N0453AVIAL -20 1 x 0.1 ml 100 mM
  ATP N0451AVIAL -20 1 x 0.1 ml 100 mM
  T7 RNA Polymerase Mix M0255AVIAL -20 1 x 0.1 ml
  GTP  N0452AVIAL -20 1 x 0.1 ml 100 mM
  10X T7 Reaction Buffer B2041AVIAL -20 1 x 0.1 ml
  Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM
  10X T7 Reaction Buffer B2041AAVIAL -20 1 x 0.5 ml 10 X
  T7 RNA Polymerase Mix M0255LVIAL -20 1 x 0.5 ml
  FLuc Control Template N0426AVIAL -20 1 x 0.01 ml 0.5 µg/µl
  ATP N0451AAVIAL -20 1 x 0.5 ml 100 mM
  GTP  N0452AAVIAL -20 1 x 0.5 ml 100 mM
  UTP  N0453AAVIAL -20 1 x 0.5 ml 100 mM
  CTP  N0454AAVIAL -20 1 x 0.5 ml 100 mM
  Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM

Properties & Usage



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FAQs
Tech Tips

  • It is important to mix each component well before setting up reactions. 

  • Make sure reactions are thoroughly mixed.

  • We recommend incubating the reactions in a dry air incubator or in a PCR machine. 

Citations
Additional Citations
  • Lee, NC., Larionov, V., Kouprina, N. (2015) Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast Nucleic Acids Res 43(8), e55.PubMedID: 25690893
  • Jaitin, DA., Kenigsberg, E., Keren-Shaul, H., Elefant, N., Paul, F., Zaretsky, I., Mildner, A., Cohen, N., Jung, S., Tanay, A. and Amit, I. (2014) Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types. Science 343, 776-779.PubMedID: 24531970
Quality Control Assay
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Specifications
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Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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