| Problem |
Possible Cause |
Solution |
Incomplete
or no
digestion |
Enzyme is inactive |
- Test enzyme on control DNA with known multiple sites
- Enzyme should be stored at -20°C. Enzymes stored at -70°C will freeze, and repeated
thaw/freeze cycles may reduce enzyme activity
|
| Reaction conditions are not optimal |
- Use recommended buffer supplied with restriction enzyme
- Follow recommendations for double digestion, or try a sequential digest
- Repeat with fresh buffer. Additives present in buffer (e.g., DTT, SAM) may degrade over
time
|
| Enzyme concentration is too low |
Some plasmids or genomic DNAs may require up to 10–20
units/µg |
| Additive is missing |
Repeat reaction setup, being sure that enzyme and/or additives (e.g.,
BSA) is added |
| DNA concentration is not optimal |
NEB recommends 1 µg of DNA in a 50 µl
reaction.
Excess DNA may result in incomplete cleavage. |
| Incubation time was too short |
Some enzymes can exhibit slower cleavage towards specific sites. In most
cases, 1–2 hours are sufficient. |
| 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 is present. Miniprep DNA is particularly susceptible to contaminants.
- Clean DNA with a spin column, resin or drop dialysis, or increase volume to dilute contaminant.
|
| Recognition site is not present |
Confirm DNA sequence |
| Cleavage is blocked by methylation |
- Some recognition sites are blocked by Dam and Dcm methylation. If site is blocked, DNA should
be passed through a dam-/dcm- strain (NEB
#C2925).
- Eukaryotic genomic DNA may be blocked by CpG methylation. This can be overcome by cloning
into a bacterial host.
- PCR products are not methylated
|
| DNA may be supercoiled |
Restriction enzymes cleave supercoiled DNA with varying efficiency.
Additional enzyme may be required. |
| Recognition site may be too close to the end of
the DNA fragment |
As a general rule, add 6 bases pairs on either side
of the recognition site for efficient cleavage |
| Site preference |
Enzyme requires two recognition sites for efficient cleavage (e.g.,
SfiI) |
Unexpected
Cleavage
Pattern |
Determine nature of pattern |
Run uncut substrate DNA alongside the digest. A partial
digest will show bands found in the uncut, whereas star activity will show bands of unexpected size.
See following suggestions for solutions. |
| DNA sample is contaminated |
Prepare a new DNA sample |
| Additional recognition sites are present in DNA |
Confirm DNA sequence |
| Star Activity |
See tips for avoiding star activity and/or use a High Fidelity Restriction
Enzyme. |
Smearing
of DNA
on
gel |
Enzyme has a high binding affinity to DNA and
will not dissociate well |
Add SDS to the gel loading dye/stop solution to a
final concentration of 0.1–0.5% to help dissociate the enzyme from the DNA |
| Nuclease contamination
|
Care should be taken to avoid cross-contamination when setting up reactions
|
| Agarose gel running conditions |
Use fresh running buffer and appropriate voltage
to avoid over heating |
