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  • HiScribe™ T7 In Vitro Transcription Kit

    This product has been discontinued and replaced by NEB# E2050,

    Discontinued Date

    06/13/2013
    Catalog #SizeConcentrationPriceQtyAdd to Cart
      
    Categories:
    Discontinued Products

    Description

    As an improved alternative, NEB now offers the T7 Quick High Yield RNA Synthesis Kit. This new kit provides the same level of performance and yield as the HiScribe T7 In Vitro Transcription Kit, but with several protocol enhancements. Learn more in the Discontinuation Notice.

    The HiScribe T7 In Vitro Transcription Kit is a system for the in vitro synthesis of large amounts of single-stranded (ss) or double-stranded (ds)RNA. This RNA can be used in numerous applications including RNA structural studies, ribozyme biochemistry, in vitro translation, RNA-protein interactions, antisense technology, aptamer discovery (SELEX), and RNA interference (RNAi) experiments. 

    The HiScribe T7 In Vitro Transcription Kit is designed for the production of RNAs that are 0.3 kb and longer (Protocol 1) or for small RNAs 50–300 nt in length (Protocol 2). 

    The kit contains sufficient reagents for synthesis of up to 3 mg RNA in 2 ml of total reaction volume. 

    DNA Template Preparation:
    The sequence to be transcribed can be cloned in a plasmid downstream of a T7 promoter (e.g. LITMUS 28i or LITMUS 38i – see companion products) or generated by PCR (Figure 1). 

    See FAQs for Plasmid and PCR Template Preparation

    Figure 1:
    In vitro transcription with different templates (A) The Litmus 28iMal Control Plasmid containing a portion of malE gene is linearized by StuI & BglII in two separate restriction digest reactions followed by inactivation of REs: Both linearized DNAs are combined and used as the template to generate double-stranded (ds)RNA or only StuI linearized DNA is used as template to generate single-stranded (ss)RNA. (B) When the target template lacks T7 sequence, it can be amplified using T7 containing primers. The PCR product is used as template to generate dsRNA. (C) When the T7 sequence is included in only one primer, the PCR product is used as template to generate ssRNA.
    Figure 2:
    In vitro transcription using either 1 µg or 2 µg of the StuI Linearized LITMUS 28iMal Control Plasmid. Transcription reactions were incubated at 42°C for either 1 or 4 hours. One μl of each 40 μl reaction was prepared in formamide containing loading buffer and loaded on a 1% agarose gel. Note: Transcribed ssRNA product (~800 nt) appears smaller than the dsDNA template. The marker lane (M) contains (non-denatured) 100 bp DNA Ladder (NEB #N3231 ).


    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Ribonucleotide Solution Mix-2025 mM of each NTP
    T7 RNA Polymerase (500 units/μl)500 units/μl
    StuI Linearized Litmus 28iMal Control Plasmid (1 mg/ml)1 mg/ml
    10X Transcription Buffer10X
    High Molecular Weight (HMW) Component Mix 20X

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. UV Absorbance: 1 A260 unit = 40 µg/ml RNA
    2. Nucleotide removal by G25 spin column is essential for accurate quantitation.

    References

    1. Schenborn, E.T. and Mierendorf, R.C. (1985). Nucl. Acids. Res.. 13
    2. Milligan, J.F. et al. (1987). Nucl. Acids. Res. 15, 8783-8798.
    3. Sampson, J.R. and Uhlenbeck, O.C. (1988). Proc. Natl. Acad. Sci. USA. 85, 1033-1037.

    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].
    1. How to prepare Plasmid Templates?
    2. How to prepare PCR Templates?
    3. Why cloned templates have to be linearized prior to transcription? Why can't I simply transcribe the circular plasmid containing my target sequence?
    4. What is the shelf life of the reagents supplied?
    5. Why is the yield for my transcription reaction lower than expected, and/or the bands are smeary?
    1. 1: In Vitro Transcription of Long Templates (> 0.3 kb) (E2030)
    2. 2: In Vitro Transcription of Short Templates (50-300 nt) (E2030)