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  • Restriction Enzyme Troubleshooting Guide

    The following guide can be used for troubleshooting restriction enzyme digestions. You may also be interested in reviewing additional tips for optimizing digestion reactions.

    • Why is My Restriction Enzyme Not Cutting DNA?

      Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.

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    • Restriction Enzyme Digest Problem: Too Many DNA Bands

      Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.

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    • Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

      Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.

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    • Restriction Enzyme Digest Protocol: Cutting Close to DNA End

      When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.

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    • Reduce Star Activity with High-Fidelity Restriction Enzymes

      NEB has engineered HF™ enzymes to eliminate star activity. Learn how, and what this means for your digests.

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    Problem Cause Solution
    Few or no
    transformants
    Restriction enzyme(s) didn’t cleave completely
    • Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
    • Use the recommended buffer supplied with the restriction enzyme
    • Clean up the DNA to remove any contaminants that may inhibit the enzyme
    • When digesting a PCR fragment, make sure to have at least 6 nucleotides between the recognition site and the end of the DNA molecule
    The digested DNA ran as a smear on an agarose gel The restriction enzyme(s) is bound to the substrate DNA
    • Lower the number of units
    • Add SDS (0.1–0.5%) to the loading buffer to dissociate the enzyme from the DNA
    Nuclease contamination
    • Use fresh, clean running buffer
    • Use a fresh agarose gel
    • Clean up the DNA
    Incomplete restriction enzyme digestion Cleavage is blocked by methylation
    • DNA isolated from a bacterial source may be blocked by Dam and Dcm methylation
    • DNA isolated from eukaryotic source may be blocked by CpG methylation
    • Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
    • If the enzyme is inhibited by Dam or Dcm methylation, grow the plasmid in a dam-/dcm- strain (NEB #C2925)
    Salt inhibition
    • Enzymes that have low activity in salt-containing buffers (NEBuffer 3.1) may be salt sensitive, so clean up the DNA prior to digestion
    • DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity. To prevent this, DNA solution should be no more than 25% of total reaction volume.
    Inhibition by PCR components
    • Clean up the PCR fragment prior to restriction digest
    Using the wrong buffer
    • Use the recommended buffer supplied with the restriction enzyme
    Too few units of enzyme used
    • Use at least 3–5 units of enzyme per μg of DNA
    Incubation time was too short
    • Increase the incubation time
    Digesting supercoiled DNA
    • Some enzymes have a lower activity on supercolied DNA. Increase the number of enzyme units in the reaction.
    Incomplete restriction enzyme digestion Presence of slow sites
    • Some enzymes can exhibit slower cleavage towards specific sites. Increase the incubation time, 1–2 hours is typically sufficient.
    Two sites required
    • Some enzymes require the presence of two recognition sites to cut efficiently
    DNA is contaminated with an inhibitor
    • Assay substrate DNA in the presence of a control DNA. Control DNA will not cleave if there is an inhibitor present. Mini prep DNA is particularly susceptible to contaminants.
    • Clean DNA with a spin column, resin or drop dialysis, or increase volume to dilute contaminant
    Extra bands in the gel If larger bands than expected are seen in the gel, this may indicate binding of the enzyme(s) to the substrate
    • Lower the number of units in the reaction
    • Add SDS (0.1–0.5%) to the loading buffer to dissociate the enzyme from the substrate
    Star activity
    • Use the recommended buffer supplied with the restriction enzyme
    • Decrease the number of enzyme units in the reaction
    • Make sure the amount of enzyme added does not exceed 10% of the total reaction volume. This ensures that the total glycerol concentration does not exceed 5% v/v
    • Decrease the incubation time. Using the minimum reaction time required for complete digestion will help prevent star activity.
    • Try using a High-Fidelity (HF) restriction enzyme. HF enzymes have been engineered for reduced star activity.
    Partial restriction enzyme digest
    • Enzymes that have low activity in salt-containing buffers (e.g., NEBuffer 3.1) may be salt sensitive. Make sure to clean up the DNA prior to digestion.
    • DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity. To prevent this, DNA solution should be no more than 25% of total reaction volume
    • Clean-up the PCR fragment prior to restriction digest
    • Use the recommended buffer supplied with the restriction enzyme
    • Use at least 3–5 units of enzyme per μg of DNA
    • Digest the DNA for 1–2 hours