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    Quick Blunting™ Kit

    Description

    The Quick Blunting Kit is used to convert DNA with incompatible 5´ or 3´ overhangs to 5´ phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors. DNA is blunted using T4 DNA polymerase (NEB #M0203) which has both 3´ → 5´ exonuclease activity and 5´ → 3´ polymerase activity. T4 Polynucleotide Kinase (NEB #M0201) is included in the enzyme mix for phosphorylation of the 5´ ends of blunt-ended DNA for subsequent ligation into a cloning vector. This kit is optimized for blunting up to 5 µg of DNA in a single reaction.

    Blunt Enzyme Mix supplied in: 100 mM KCl, 10 mM Tris-HCl(pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% Glycerol.

    1X Blunting Buffer:
    100 mM Tris-HCl
    50 mM NaCl
    10 mM MgCl2
    0.025% Triton X-100
    5 mM dithiothreitol
    pH 7.5 at 25°C

    Highlights

    Fast — Restriction enzyme digested DNA is blunted in less than 30 minutes
    Convenient — Reactions are performed at room temperature in a ready-to-use mix
    Flexible —Suitable for restriction enzyme digested DNA, sheared or nebulized DNA or PCR product

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    10X Blunting Buffer10X
    Deoxynucleotide Solution Mix (1 mM)1 mM
    Blunting Enzyme Mix20 reactions

    Advantages and Features

    Applications

    Prepare sheared, nebulized or restriction enzyme digested DNA for blunt-ended ligation into a plasmid, cosmid, fosmid or BAC vectorbr/ Prepare PCR products for efficient blunt-end cloning

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
    2. Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1,2,3 and 4, as well as BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 where the total yield is about 50% of optimum.
    3. ATP is not necessary for T4 Polynucleotide Kinase activity in the kit. The dATP and dTTP in the dNTP mix act as phosphate donors.
    4. The blunted reaction must be purified prior to phosphatase treatment by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.

    FAQs

    1. Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit?
    2. Can I use regular ligase to ligate my blunted product?
    3. I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction?
    4. Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product?
    5. I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest?
    6. I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work?
    7. I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work?

    Protocols

    1. Blunting protocol for NEB PCR Cloning Kit (E1202)

    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.

    Selection Tools

    Troubleshooting Guides

    Material Safety Data Sheets

    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.