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  • T7 High Yield RNA Synthesis Kit

    Description

    The 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.  Small kits include 0.1 ml, and large kits include 0.5 ml of each ATP, GTP, UTP and CTP.

    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. 
    • Cap Analogs: NEB #S1411, #S1405, #S1406 and #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 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
    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
    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 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 T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ATP 100 mM
    GTP 100 mM
    UTP 100 mM
    CTP 100 mM
    10X T7 Reaction Buffer10X
    FLuc Control Template0.5 μg/μl
    T7 RNA Polymerase Mix

    Properties and Usage

    Storage Temperature

    -20°C

    Troubleshooting

    Troubleshooting

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. DNA Template Preparation (E2040)
    2. RNA Synthesis with Modified Nucleotides (E2040)
    3. Purification of Synthesized RNA (E2040)
    4. Standard RNA Synthesis (E2040)
    5. Capped RNA Synthesis (E2040)
    6. High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
    7. Evaluation of Reaction Products (E2040)

    Citations

    • 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

    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.