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.
Videos
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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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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 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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Time-Saver Protocol for Restriction Enzyme Digests
Need a protocol to digest quickly and completely? Try this protocol for Time-Saver™ qualified enzymes from NEB.
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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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Standard Protocol for Restriction Enzyme Digests
Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.
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CutSmart™ Restriction Enzyme Buffer
More than 210 of NEB's restriction enzymes are 100% active in a single buffer. Learn more about CutSmart™ Buffer and why it matters to you.
Problem | Cause | Solution |
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Few or no transformants |
Restriction enzyme(s) didn’t cleave completely |
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The digested DNA ran as a smear on an agarose gel | The restriction enzyme(s) is bound to the substrate DNA |
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Nuclease contamination |
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Incomplete restriction enzyme digestion | Cleavage is blocked by methylation |
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Salt inhibition |
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Inhibition by PCR components |
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Using the wrong buffer |
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Too few units of enzyme used |
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Incubation time was too short |
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Digesting supercoiled DNA |
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Incomplete restriction enzyme digestion | Presence of slow sites |
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Two sites required |
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DNA is contaminated with an inhibitor |
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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 |
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Star activity |
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Partial restriction enzyme digest |
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1Monarch Kits (NEB #T1010, NEB #T1020, and NEB #T1030) use columns that have been designed to minimize salt carry over into the eluted DNA, so using them can minimize this issue.