Troubleshooting Common Issues with Restriction Digestion Reactions: How to Avoid or Solve Problems

The most common issues when performing digestion reactions are caused by unexpected cleavage patterns. In this article, we have highlighted the most recurrent problems and probable causes to help you resolve your hurdles.

Below is the guide of what issues this article will be covering.

Avoid storing enzymes in frost-free freezers that undergo temperature fluctuations. It is recommended to keep the enzymes in a cold rack in the freezer, as this helps to stabilize the storage temperature.
 

If there are no issues with digesting the control DNA, there may be something else wrong with your reaction setup.

Solution check list

• ☑  Verify that you are using the recommended reaction buffer, including any additives specified in the product support material. Avoid improper buffers by using FastDigest restriction enzymes, which all work in the same buffer
• ☑  Make sure you are using molecular biology–grade water.
• ☑  Ensure that the restriction enzyme is the final component added to the reaction mix, and that the final glycerol content is below 5%, so that the enzyme volume represents less than one tenth of the total reaction volume. 
• ☑  Double check the optimal reaction temperature for the enzymes being used, and control for evaporation during incubation as an increased salt concentration in the buffer can inhibit enzyme performance.
• ☑  Optimal DNA concentration is between 20–100 ng/µL in the final reaction mixture. 
• ☑  DNA substrate is free of contaminants or reaction components like SDS, EDTA, protein, salts, and ethanol.
   

Several endogenous methylases site-specifically methylate adenine (DAM) or cytosine (DCM) residues and can affect enzyme activity (Figure 2).

For example, methylation by deoxyadenosine methylase (DAM methylation) occurs normally in E. coli at GATC sequences. This sequence overlaps with the recognition sites of some enzymes, like BamHI and BclI. In this case, BamHI cuts the DNA in the presence or absence of methylation, while BclI cannot cleave methylated DNA. To overcome this restriction, you can transform your plasmid DNA into a dam-minus, dcm-minus strain, such as E. coli GM2163. These methylation minus strains do not interfere with methylation (Figure 3). ​

To approach this issue, first check what type of sample you are dealing with: PCR fragments or plasmids.

Because recognition sites are often introduced at the ends of PCR fragments and/or primers, it is important to understand how many bases flanking a site are needed for optimal cleavage. 

Enzyme suppliers often provide tables that illustrate how many bases from the end of a recognition site should be present for optimal activity. 

For plasmid DNA:

If you are trying to perform a double digest with two enzymes in the multiple cloning site, efficient cleavage may be difficult if the two recognition sites are too close together. One enzyme may physically block access of the second enzyme to its respective site.

Inefficient cleavage is also related to the previously described proximity of the recognition site to DNA ends. After one enzyme cuts, there may not be enough bases flanking the second site for the second enzyme to bind and effect cleavage.

Due to the reasons above, consider doing a sequential digestion. Before you set up the reaction, determine which enzyme is more effective at cutting close to the end of a DNA fragment, and use that enzyme second.

For example, XbaI and SalI are next to each other in the pUC19 multiple cloning site (MCS) (Figure 6). If you cut with XbaI first, SalI would only cut with up to 20% efficiency.

However, if you cut with SalI first, XbaI can cut near the end of the strand, with up to 100% efficiency. For this reason, you should perform sequential reactions and digest with SalI followed by XbaI to  prepare the plasmid for cloning (Figure 7).

If your probable cause is insufficient incubation time, you should gradually increase incubation time. Longer incubation times are often used to allow a reaction completion with fewer units of enzyme.
 

In general, it is recommended to use 3 to 5 units of enzyme per microgram of DNA. Consult with your supplier and product support materials to obtain recommended enzyme concentration. If digesting supercoiled DNA, increase enzyme units in the reaction.
 

Dealing with contaminated templates is common. Use spin column or PCR clean up kit to remove contaminants. The volume of DNA cannot exceed 25% of the total volume of the digestion reaction.
 

• Star activity (off-target cleavage)
• High glycerol concentration (greater than 5% in the final reaction)
• High enzyme:DNA ratio, or overdigestion
• High pH or low ionic strength
• Presence of organic solvents such as DMSO or ethanol
• Use of a divalent cation other than magnesium in the reaction mix
• Inclusion of other non-optimal buffer conditions
• Prolonged incubation, such as overnight digestion

If you’re experiencing star activity or any of the causes above, here are all the key factors/areas you should check: 

Choose an enzyme supplier that has addressed star activity as follows: 

• ☑  Optimizes their enzymes and buffers to minimize star activity, 
• ☑  Offers isoschizomers with low or no intrinsic star activity, and 
• ☑  Offers engineered or modified enzymes to eliminate star activity. 
• ☑  Follow the recommendations provided with each enzyme for optimal activity including the use of the correct buffer, enzyme amount, and reaction time for the enzyme. 

Most enzymes will not exhibit star activity when used under recommended conditions in optimal buffers. However, under suboptimal or extreme conditions, star activity may occur. Here’s a quick guide on How to Recognize Star Activity.

For example, incomplete digestion results in additional bands above the expected bands on a gel. These bands disappear when the incubation time or amount of enzyme is increased, as seen when comparing sample in lanes 2 and 3 to the completely digested sample in lane 4. 

Star activity, as seen in lanes 5 and 6, results in additional bands below the smallest expected size. These bands will generally become more intense with increasing enzyme dose or time, while the expected bands become less intense (Figure 8). 

Most commercial enzymes are formulated with glycerol for stability and to prevent freezing at –20°C. 
 

Gel-shift is the result of another enzyme attribute and can result in an unexpected banding pattern when viewing digested samples on a gel. It is typically more apparent when high enzyme doses are used and can have minor or significant impact on visualizing samples (Figure 9A).

A second method to reduce gel-shift is to add SDS into the loading buffer prior to loading on the gel. These methods will denature the enzyme, releasing it from the DNA fragment (Figure 9B).

The restriction enzyme tube or reaction buffer tube may be contaminated with a second enzyme. This can happen where the same reaction buffer is used for multiple different enzymes.

• Try a fresh tube of enzyme or reaction buffer. 
• Check for contamination by using a fresh DNA preparation. 

In rare cases, it may be possible that there are unexpected recognition sites in the substrate DNA.
You can check for mutations that may have been introduced during PCR amplification. There is also potential to generate new restriction sites after ligation of DNA fragments.