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.

We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer™ 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We anticipate that this switch may take as long as 6 months to complete. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Find more details at www.neb.com/BSA-free.

During this transition period, you may receive product with BSA or rAlbumin-containing buffers. NEB has rigorously tested both and has not seen any difference in enzyme performance when using either buffer. Either buffer can be used with your enzyme. All website content will be switched in April to reflect the changes, although you may not receive the new buffer with your product immediately.

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 or use Gel Loading Dye, Purple (6X) (NEB #B7024)
Nuclease contamination
  • Use fresh, clean running buffer
  • Use a fresh agarose gel
  • Clean up the DNA (NEB #T1030)
Incomplete restriction enzyme digestion Cleavage is blocked by methylation
  • Check the methylation sensitivity of the enzyme(s) to determine if the enzyme is blocked by methylation of the recognition sequence
  • DNA isolated from a bacterial source may be blocked by Dam and Dcm methylation
  • If the enzyme is inhibited by Dam or Dcm methylation, grow the plasmid in a dam-/dcm- strain (NEB #C2925)
  • DNA isolated from eukaryotic source may be blocked by CpG methylation
Salt inhibition
  • Enzymes that have low activity in salt-containing buffers (NEBuffer r3.1) may be salt sensitive, so clean up the DNA (NEB #T1030) prior to digestion
  • DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity.1 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 (NEB #T1030)
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 (NEB #T1030) 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 r3.1) may be salt sensitive. Make sure to clean up the DNA (NEB #T1030) prior to digestion.
  • DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity.1 To prevent this, DNA solution should be no more than 25% of total reaction volume.
  • Clean-up the PCR fragment prior to restriction digest (NEB #T1030)
  • Use the recommended buffer supplied with the restriction enzyme
  • Use at least 5–10 units of enzyme per μg of DNA
  • Digest the DNA for 1–2 hours


1
Monarch 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.