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  • Optimizing Restriction Endonuclease Reactions When Using RE-Mix® Restriction Enzyme Master Mix

    Restriction Digest Using A RE-Mix® Restriction Enzyme Master Mix

    RE-Mix Master Mix 2 µl
    DNA 1 µg
    Total Reaction Volume 20 µl
    Incubation Time 15 minutes
    Incubation Temperature 37°

    Enzyme

    • Keep on ice when not in the freezer
    • Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
    • In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.
    • RE-Mix Master Mixes should be used at 1X concentration.

    DNA

    Buffer

    • RE-Mix Master Mixes already include buffer and BSA so no additional buffer is supplied.

    Reaction Volume

    • A 20 µl reaction volume is recommended for digestion with a RE-Mix Master Mix.

     Incubation Time

    • The recommended incubation time with a RE-Mix Master Mix is 15 minutes.

    Stopping a Reaction

    • The RE-Mix Master Mix includes a density agent and dye and does not require addition of a stop solution.
    • When further manipulation of DNA is required:
    • Heat inactivation can be used.
    • Remove enzyme by using a spin column or phenol/chloroform extraction.

    Storage

    • RE-Mix Master Mixes should be stored at -20°C.

    Stability

    • All enzymes are assayed for activity every 4 months. The expiration date is found on the label.
    • Exposure to temperatures above -20°C should be minimized whenever possible.

    Control Reactions

    If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions:
    • Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability
    • If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.

    Re-Mix® is a registered trademark of New England Biolabs, Inc.