This troubleshooting guide addresses common problems that may be encountered while
using restriction enzymes. If problems persist contact Promega Technical Services at
techserv@promega.com or 800-356-9526 (U.S. and Canada
only) or your local Promega Branch
Office or Distributor.
| Problem:
No cleavage |
| Probable Cause |
Comments |
| Dirty template DNA |
Clean up the substrate DNA using the Wizard®
DNA Clean-Up System(a) (Cat.# A7280).
Alternatively, phenol/chloroform extraction followed by ethanol precipitation can be used
to purify the DNA substrate. A discussion of substrate quality and
considerations can be found in the Substrate Considerations section
of this guide. |
| Presence of inhibitors |
Enzyme inhibitors in the substrate DNA solution (e.g.,
SDS, phenol, EDTA, chloroform, ethanol, CsCl, high salt, or plasticizers from
microcentrifuge tubes) can be removed using the Wizard® DNA Clean-Up System.
Alternatively, phenol/chloroform extraction and/or ethanol precipitation can be used to
remove inhibitors from the DNA preparation. |
| DNA methylated (e.g. dam, dcm) |
Check Table 3.5 for information on the sensitivity of the
restriction enzyme to the methylation state of the substrate. For plasmid
DNA, eliminate methylation by passaging the DNA through a bacterial host that is deficient
in the interfering methylase. For example, E. coli JM110, which lacks both dam
and dcm activity.
Digest the DNA using an isoschizomer that is insensitive to methylation. Consult
Table 3.6 for
isoschizomer enzyme pairs that differ in their ability to cut methylated DNA. |
| DNA unmethylated |
Some enzymes require methylation of their target
sites. For example, Dpn I requires N6-methylation of the adenine residue
for activity. See Table 3.5 for further
information on the effect of site-specific methylation on Promega restriction enzymes. |
| Inactive enzyme |
Test the enzyme on substrate DNA that has been
digested successfully in the past or test the enzyme with the DNA substrate used for
determination of the enzyme unit activity. Usually this is Lambda DNA (Cat.# D1501). |
| Suboptimal reaction conditions |
Consult the Promega Product Information sheet provided
with the enzyme for recommended reaction conditions. Suggested reaction conditions can be
found in the Standard Reactions section of this guide. |
| Incorrect sequence information |
Double check sequence information to confirm the
number and location of enzyme recognition sites. |
| Problem:
Partial Cleavage. |
| Probable Cause |
Comments |
| Dirty template DNA |
Clean up the substrate DNA using the Wizard®
DNA Clean-Up System (Cat.#
A7280). Alternatively, phenol/chloroform extraction followed by ethanol precipitation
can be used to purify the DNA substrate. A discussion of substrate
quality and considerations can be found in the Substrate Considerations
section of this guide. |
| Loss of restriction enzyme activity |
Digest substrate DNA with several other restriction
enzymes to ensure that impurities in the digest are not interfering with enzyme activity.
Alternatively, the enzyme activity can be tested using the unit activity assay conditions.
The Promega Product Information sheet supplied with the enzyme contains information on the
unit activity assay conditions specific for that enzyme. See the comments
under the problem Enzyme activity lower than expected for more
possible causes and solutions for low restriction enzyme activity. |
| Presence of enzyme inhibitors |
Enzyme inhibitors in the substrate DNA (e.g., SDS,
phenol, EDTA, chloroform, ethanol, CsCl, high salt, or plasticizers from microcentrifuge
tubes) can be removed using the Wizard® DNA Clean-Up System (Cat.# A7280).
Alternatively, phenol/chloroform extraction and/or ethanol precipitation can be used to
remove inhibitors from the DNA preparation. |
| Improper reaction conditions |
Check that proper reaction conditions were used
including the optimal buffer, temperature and amount of enzyme. Suggested reaction
conditions can be found in the Standard Reactions section of this
guide or on the Promega Product Information sheet provided with each restriction enzyme. |
| Restriction enzyme not completely mixed into reaction |
Add the restriction enzyme to the digest last and mix
gently. Ensure that the enzyme is mixed thoroughly into the reaction but do not vortex. |
| Loss of restriction enzyme activity upon dilution prior
to use |
If possible, do not dilute the enzyme prior to use. If
the enzyme must be diluted, use the recommended storage buffer for that enzyme. If used
immediately, enzyme can be diluted in Reaction Buffer containing 0.5mg/ml Acetylated BSA.
Enzymes diluted into the reaction buffer do not store well. Never dilute
the enzyme directly in water. Mix gently, do not vortex. |
| Loss of restriction enzyme activity upon addition to
digest |
Enzyme has lost activity upon dilution into reaction.
Use optimum restriction enzyme buffer supplemented with 0.1mg/ml Acetylated BSA to
stabilize enzyme in the reaction. |
| DNA concentration too high |
Reduce DNA concentration or use multiple reactions.
Viscous DNA solutions can inhibit enzyme digestions. |
| DNA concentration too low |
Sample DNA concentration is below the Km of
the restriction enzyme. Add more DNA to the reaction. |
| Annealed DNA ends (e.g., lambda DNA) |
The ends of some DNA substrates such as the cos
ends of lambda may re-anneal during digestion. This can give the appearance that digestion
is incomplete. Heat the DNA at 65°C for 5 minutes prior to gel electrophoresis to melt
ends that have annealed. The presence of restriction enzyme buffer is important while
heating as this will prevent small DNA fragments from melting into single-strands. |
| Denaturation of restriction enzyme |
Many restriction enzymes can be inactivated by heat.
Also, avoid vortexing dilutions or reactions containing restriction enzymes. |
| DNA substrate is supercoiled |
Supercoiled DNA generally requires more units of
enzyme than linear DNA for complete digestion. The unit activity of restriction enzymes is
determined using linear DNA templates. One unit of restriction enzyme may cut one
microgram of linear DNA in one hour, but this may not be true of supercoiled DNA. See Digestion of Supercoiled Plasmid DNA for further information.
Alternatively, try using five units of restriction enzyme per microgram of supercoiled DNA
for digestion in one hour. Another option is to linearize the DNA with
an enzyme that is not resistant to supercoiling, then digest with the enzyme of choice.
Alternatively, relax the DNA with topoisomerase, then digest with the restriction enzyme. |
| Substrate DNA has many restriction sites per unit of
mass |
The Promega Product Information sheet supplied with
each enzyme lists the substrate DNA used in the unit activity assay and how many cut sites
the substrate has for that enzyme. While one unit of enzyme will cut 1µg of the activity
assay substrate to completion in one hour, DNA with many more cut sites per microgram will
require more units of enzyme for complete digestion in one hour. See Substrate
Considerations for further information. The optimum amount of enzyme should be
determined for each substrate. |
| Problem: Enzyme
activity lower than expected. |
| Probable Cause |
Comments |
| Suboptimal reaction conditions |
Consult the Promega Product Information sheet provided
with the enzyme for recommended reaction conditions. Suggested reaction conditions also
can be found in the Standard Reactions section of this guide. |
| Incorrect storage or handling of enzyme |
Store all restriction enzymes at -20°C in a non-frost
free freezer. Remove the enzyme just prior to use and keep on ice. Return the enzyme to
the freezer as soon as possible. Do not vortex the enzyme or the reaction mix containing
the enzyme. Instead, mix by gentle pipetting. Avoid air bubbles. |
| Enzyme stored diluted |
It is best to store the enzymes as supplied in
concentrated form. If the enzyme must be diluted, use the recommended enzyme storage
buffer. If used immediately, enzyme can be diluted in Reaction Buffer containing 0.5mg/ml
BSA. Enzymes diluted in Reaction Buffer do not store well. |
| Incorrect dilution of enzyme |
If enzyme is diluted just prior to use, dilute the
enzyme in the recommended 1X Reaction Buffer supplemented with 0.5mg/ml Acetylated BSA.
Never dilute enzyme directly in water. Check the dilution factor used to ensure that the
enzyme concentration is correct. Use the enzyme as soon as possible after dilution. |
| Pipetting error |
Use a positive displacement pipet for viscous
solutions such as concentrated DNA and enzyme storage buffer, which contains 50% glycerol.
|
| Glycerol inhibition |
Inhibition of the reaction may occur if the volume of
enzyme added is greater than 10% of the total reaction volume. |
| Reaction temperature suboptimal |
Check for optimal reaction temperature on the Promega
Product Information sheet supplied with the enzyme. See Table 3.1 for a listing of optimal temeratures for
all Promega restriction enzymes. |
| DNA substrate is supercoiled |
Supercoiled DNA generally requires more units of
enzyme than linear DNA for complete digestion. The unit activity of restriction enzymes is
determined using linear DNA substrates. One unit of restriction enzyme may cut one
microgram of linear DNA in one hour, but this may not be true of supercoiled DNA. See Digestion of Supercoiled Plasmid DNA for
further information. Alternatively, try using five units of restriction enzyme per
microgram of supercoiled DNA for digestion in one hour. |
| Substrate DNA has many restriction sites per unit of
mass |
The Promega Product Information sheet supplied with
each enzyme lists the substrate DNA used in the unit activity assay and how many cut sites
the substrate has for that enzyme. While one unit of enzyme will cut 1µg of the activity
assay substrate to completion in one hour, DNA with many more cut sites per microgram will
require more units of enzyme for complete digestion in the same time. See Substrate Considerations for further information. The optimum amount of
enzyme should be determined for each substrate. Guidelines for Digestion
of High Molecular Weight DNA can be found elsewhere in this guide. |
| Suboptimal reaction conditions |
Consult the Promega Product Information sheet provided
with the enzyme for recommended reaction conditions. Suggested reaction conditions can be
found in the Standard Reactions section of this guide. |
| Problem: Greater
than expected number of DNA fragments. |
| Probable Cause |
Comments |
| Star activity |
Star activity or relaxed specificity of the
restriction enzyme for its cognate sequence is caused by suboptimal digestion conditions.
Common causes of star activity include the use of excess enzyme (generally
>100units/µg), excess glycerol (>5% v/v), the presence of manganese or other
divalent cation instead of magnesium, or nonoptimal NaCl concentrations. Extremes of pH
(especially >pH 8.0) and the presence of DMSO, ethanol or other organic solvents are
also causes of star activity. For more information, see Star
Activity. To avoid star activity, use the recommended digestion
conditions for the enzyme and avoid using DNA substrates that may be contaminated with
salts or solvents. Conditions for setting up a restriction enzyme digest can be found in
the Standard Reactions section of this
guide and on the Promega Product Information sheet supplied with each enzyme. |
| Sample contaminated with another DNA |
Confirm the activity of the restriction enzyme by
testing either the substrate used for the unit activity assay (generally lambda DNA)
or another substrate known to contain a single DNA species. If the digestion pattern is
correct for these substrates then the extra bands present may be due to another DNA
contaminating the reaction. Test the enzyme, reaction buffers and gel loading buffer for
DNA contamination. |
| Presence of a second restriction enzyme |
Detect a second activity by repeating the unit
activity assay or test a known DNA substrate with a defined number of cut sites for the
enzyme. |
| Volume of reaction decreased during long digestion |
During extended digestions the volume of the digest
may decrease, especially if a thermophilic restriction enzyme is used. A reduction in the
volume of the reaction may lead to star activity by concentrating the glycerol, enzyme,
salt, or any contaminants in the reaction. For long digestions or digestions at elevated
temperatures, add mineral oil to the surface, decrease the incubation time of the reaction
or perform the reaction in an incubator. |
| Incorrect sequence information |
Double check sequence information to confirm the
number and location of enzyme recognition sites. |
| Problem: No
DNA observed after digestion. |
| Probable Cause |
Comments |
| Concentration of DNA substrate incorrect |
Determine the concentration of the DNA used in the
digestion by spectrophotometry or gel electrophoresis prior to digestion. Excessive
RNA or salt (e.g., guanidine) contamination of the DNA sample will increase the absorbance
at 260nm, leading to an artificially high determination of concentration. Confirm DNA
concentration by electrophoresis. If necessary, treat the sample with RNase or
ethanol-precipitate to remove RNA or salts, respectively. |
| Nuclease contamination from bacterial host |
If the DNA was isolated from an endA(+)
bacterial strain, it may be contaminated with endonuclease I. If so, when magnesium is
present (as in a restriction enzyme digest), the endonuclease will be activated and the
DNA substrate digested. If possible use an endA(-) strain for propagation of the
DNA substrate. The Wizard® Plus DNA purification products(a) can be used with endA(+) strains to produce
endonuclease-free DNA. Consult the Wizard® Plus SV Minipreps DNA
Purification System(a) Technical Bulletin, #TB225 for more information. Alternatively
phenol/chloroform extract the DNA sample before digestion to eliminate endonuclease I
contamination. More information on endonuclease I contamination can be
found in Promega Notes 53, 12. |
| Nuclease contamination from reagents |
Test individual reaction components for nuclease
contamination. Bacterial and fungal contamination is often the source of nucleases. |
| Problem: A
smear of DNA is observed after digestion. |
| Probable Cause |
Comments |
| Nuclease contamination from bacterial host or nuclease
contamination from reagents |
Complete nonspecific digestion will result in
disappearance of the DNA substrate. Partial digestion will result in a smear of DNA being
observed from a point at the estimated size of the DNA substrate to the bottom of the gel. |
