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9: Ligation of Products

  • Page ID
    79498
    • Nathan Reyna, Ruth Plymale, & Kristen Johnson
    • Ouachita Babtist University & University of New Hampshire

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    Ligation of Products

    Joining of 2 DNA molecules catalyzed by DNA ligase

    This process forms a phosphodiester bond between the 3’ hydroxyl and 5’ phosphate of adjacent DNA strands, creating recombinant DNA. (NEB)

    Video example of performing a ligation

    Materials Storage Temperature
    Water Room Temp
    10X T4 DNA Ligation Buffer -20°C
    Linearized Plasmid Backbone
    (Amp,Chlor,Kan,Tet)
    -20°C
    Double Digested DNA insert fragments -20°C
    T4 DNA Ligase -20°C

    TIPS BEFORE STARTING:

    An equal (1:1) molar quantity of each insert is best. However, 1 \(\mu L\) of each digested insert is commonly used for the ligation and gets consistent results. If your parts are drastically different in size, you may want to adjust for molar quantity. (If you are not getting good results/colonies upon transformation, start looking at changing the molar ratio of your inserts. Use NEBioCalculator to calculate molar ratios.)

    Procedure

    1. Thaw 10X T4 DNA Ligation Buffer at room temperature and then store it on ice until needed. Leave DNA ligase (enzyme) in the freezer block until immediately before it is needed; always keep cold!

    2. Always add water and buffer first. Buffer must be fully thawed and mixed before use:

    5 \(\mu L\) of Water (water up to 10ul, if insert volumes are adjusted)
    1 \(\mu L\) of 10X DNA ligation buffer
    1 \(\mu L\) (25ng) linearized plasmid (destination) backbone
    1 \(\mu L\) of insert A (digested with appropriate enzymes / denatured)
    1 \(\mu L\) of insert B (digested with appropriate enzymes / denatured)
    1 \(\mu L\) of T4 DNA ligase (actual enzyme)

    3. Incubate at room temperature for 1hr to 2hr. Ligations can go overnight or longer if stored at 4 °C (refrigerated). This works the best.

    4. Heat inactivate the reaction at 65°C (or 80°C) for 10 minutes. (Optional but company recommended.)

    5. Samples can be stored at -20°C or used immediately in the Zippy transformation protocol. It is best if you immediately transform the 5 \(\mu L\) of your reaction into a tube of zippy cells. (Ligation buffer can reduce transformation efficiency. Using more than 5ul may actual decrease transformation efficiency)


    This page titled 9: Ligation of Products is shared under a not declared license and was authored, remixed, and/or curated by Nathan Reyna, Ruth Plymale, & Kristen Johnson.

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