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  • Universal miRNA Cloning Linker

    Description

    This 5´ adenylated, 3´ blocked oligodeoxynucleotide can be used for cloning short RNAs according to the procedure of Bartel (1). RNA ligase recognizes the "activated" adenylated oligo and covalently ligates its 5´ end to the 3´ OH of a second single stranded sequence in the absence of ATP. In a mixture of nucleic acids use of the 5´ adenylated, 3´ blocked oligo with RNA ligase (w/o ATP) results in ligation of the target oligo only. 

    Linker/RNA Ligation Protocol: The Universal miRNA cloning linker has a 3´ blocking group (amine) to prevent self ligation, circularization and ligation to RNA at the 5´ end. It also contains the BanI restriction site compatible with the miRNA cloning protocol used by Bartel (1).

    For ligation, resuspend the lyophilized oligonucleotide in RNAse-free water to 100 μM stock concentration. (A resuspension volume of 8.6 μl will give a 0.58 μg/μl or 100 μM stock solution). Ligations are carried out with T4 RNA Ligase I in the absence of ATP. Approximately 1 μg is typically used per reaction. See reference 1 for a detailed protocol.

    Sequence

    5´ rAppCTGTAGGCACCATCAAT–NH2 3´

    Advantages and Features

    Applications

    • small RNA cloning (1,2)
    • miRNA library construction (1,2)

    Properties and Usage

    Storage Temperature

    -20°C

    Suggested Working Concentration

    100μM

    Recommended Resuspension Volume

    8.6 μl

    Quality Control

    Quality Assurance Statement

    • This linker was tested for reactivity with T4 RNA Ligase (1,2). Its identity was confirmed by MALDI-TOF Mass Spectrometry.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Physical Purity (HPLC):
      The purity of the product is determined by HPLC analysis.

    References

    1. Lau et al. (2001). Science. 294, 858-6.
    2. Pfeffer, S., Lagos-Quintana, M. and Tuschl, T. (2005). Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (26.4.14).

    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.

    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.

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