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  • Quick Ligation™ Kit

    Description

    The Quick Ligation™ Kit enables ligation of cohesive end or blunt end DNA fragments in 5 minutes at room temperature . (25°C )

    Ligation Time Course: LITMUS 28 vector was cut with either EcoRV (blunt) or HindIII (cohesive), treated with calf intestinal phosphatase and gel purified. Blunt inserts from a HaeIII digest of ΦX174 DNA and cohesive inserts from a HindIII digest of λ DNA were ligated into the respective vectors at a 3:1 insert:vector ratio using the Quick Ligation Kit. Ligation products were transformed into chemically competent E. coli DH-5α cells and grown overnight on LB-amp plates at 37°C.

    1X Quick Ligation Reaction Buffer:
    66mM Tris-HCL
    10mM MgCl2
    1mM Dithiothreitol
    1mM ATP
    7.5% Polethylene glycol (PEG6000)
    pH 7.6 @ 25°C

    Highlights

    Fast - 5 minutes for cohesive or blunt ends
    Convenient - ligation performed at room temperature
    Flexible - suitable for all common ligation reactions

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Quick Ligation Reaction Buffer2X
    Quick T4 DNA Ligase-20

    Advantages and Features

    Applications

    Cloning into vectors
    Library construction
    TA cloning
    Linker ligation
    Recirculization of linear DNA

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. Some of the most critical parameters which should be controlled to ensure successful ligation and transformation are addressed below. 
      Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates to the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes. 
      Electroporation: Electroporation can increase transformation efficiency by several logs. Before using the products of a Quick Ligation reaction for electrotransformation, it is necessary to reduce the PEG concentration. We recommend a spin column purification. 
      DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q™ water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the volume of DNA and insert should be 10 μl before adding 2X Quick Ligation Buffer. For DNA volumes greater than 10 μl, increase the volume of 2X Quick Ligation Buffer such that it remains 50% of the reaction and correspondingly increase the volume of ligase. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. 
      Time: Most ligations performed using the Quick Ligation™ Kit reach an end point at 5 minutes or less at 25°C. Incubation beyond this time provides no additional benefit. In fact, transformation efficiency starts to decrease after 1 hour and is reduced by up to 75% if the reaction is allowed to go overnight at 25°C. 
      Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

    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.
    1. What factors can cause incomplete ligation and/or transformation using the Quick Ligation Kit?
    2. Why is my transformation not working and what control reactions should be run?
    3. What is the concentration of T4 DNA Ligase provided in the Quick Ligation Kit?
    4. Can conventional T4 DNA Ligase (400,000 units/ml) be used with the Quick Ligation buffer?
    5. How to calculate the molarity of ends?
    1. Quick Ligation Protocol (M2200)
    2. Transformation Protocol

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Application Notes

    Citations

    • Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. (2013) Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research DOI: doi:10.1093/nar/gkt601