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
|
