Optimize Your DNA Ligations

Molecular cloning is the process used for taking recombinant DNA (referred to as an insert) and placing it into a DNA vector (i.e., plasmid) where it can be replicated and expressed. This process involves multiple steps (such as copying the DNA, cutting out the gene of interest, and pasting the gene into the DNA vector). The final step, ligation (aka the pasting step) is used to seal the insert into the vector. Ligation works by using a phosphodiester bond to connect the sugar backbone of the double-stranded DNA insert with the sugar backbone of the double-stranded DNA vector. This is typically done by using T4 DNA ligase.

T4 DNA ligase is an enzyme that helps create the formation of a phosphodiester bond between the 3'-hydroxyl end of a double-stranded DNA fragment and the 5'-phosphate end of the same or another DNA fragment (Figure 1). T4 DNA ligase can catalyze a reaction between blunt-end (no overhangs) or sticky end (3' or 5' complementary single-stranded overhangs) DNA fragments. T4 DNA ligase activity requires Mg2+ and ATP to work, and requires 5'-phosphorylation of one or both fragments.

There are many variables that are necessary to obtain maximum ligation efficiency and accuracy during cloning. As such, the ligation step can fail for numerous reasons, including:

• Problems related to the ligase enzyme itself (concentration, enzyme stability)
• Issues that occur before the addition of T4 DNA ligase (for example, presence of inhibitors including salts, EDTA, proteins, phenol, ethanol, and dATP)
• Everything else (molar insert to vector ratio, temperature, buffer composition, etc.)

To help your cloning experiment move forward, here are seven tips to help when you encounter DNA ligation failures:

Different cloning strategies can be used depending on the type of ends on the DNA fragments you are using (Figure 2).

The first three panels in Figure 2 illustrate strategies for cloning fragments with distinct sticky ends. These fragments are created when restriction enzymes cut in different places within the double-stranded DNA, resulting in overhangs (unpaired nucleotides). These “sticky ends” can be very helpful and are something that should be considered first when designing your cloning strategy. For example, if you want to clone your DNA insert so that it is read in a specific direction or orientation (also known as directional cloning), sticky end ligation is the method of choice since the created overhangs will only ligate in a specific orientation. Another reason for using sticky ends is to increase your ligation efficiency. When compared with the alternative (blunt-end ligation), sticky end ligation is more efficient due to the compatible overhangs that assist with the ligase reaction.

However, it is not always possible to use restriction enzymes that cut in different places to create sticky ends. For those cases, you would choose a blunt-end cloning strategy to ligate your DNA insert into your vector as illustrated in the fourth panel of Figure 2. For this approach, you can either choose a restriction enzyme that will generate blunt ends, or you can generate sticky ends and remove the overhangs by using an end repair kit. Since blunt-end ligation is less efficient than sticky end ligation, this approach will require additional optimization and planning. Using a higher DNA insert to vector ratio is recommended to help ensure ligation of your DNA insert into your vector while preventing vector re-circulization (ligation of your vector without the DNA insert). To further help prevent vector re-circulization, treating the vector with DNA phosphatase helps to remove the 5’-end phosphate groups from the vector before the ligation step.

If the DNA inserts you are ligating have blunt ends, the inserts must be 5'-phosphorylated at both ends in order for ligation to occur. If the DNA insert is generated from restriction enzyme digestion, the 5'-phosphate group is already present.

However, if your DNA insert is a PCR product created with a proofreading DNA polymerase, your DNA insert will not have a 5’-phosphate group. Therefore, a phosphate group must be added using T4 polynucleotide kinase (T4PNK).

When your DNA insert is a PCR product created with a Taq-like DNA polymerase, the resulting PCR product will have deoxyadenosine (dA) protruding ends since Taq DNA polymerases add a single dA to the 3´ ends of PCR products. DNA inserts that have dA ends can be ligated into vectors with complementary overhangs (this is a technique known as TA cloning). If, however, the vector you are using with TA cloning contains blunt ends, then your DNA insert also must be blunted (overhangs removed) before ligation.

If the DNA inserts you are ligating have sticky ends, they not only need the 5’-phosphates, but you will also need to make sure that the overhangs (sticky ends) on the inserts are complementary to your vector. If your overhangs are not complementary (ragged ends), your insert will not “paste” to your vector and your ligation will fail. Ragged ends can occur due to incomplete restriction enzyme digestion, elimination of your overhangs by a DNA polymerase that was not removed during purification of your insert, or by contaminating nucleases that might be present in the enzymes used to create the ends of your DNA insert. The best way to determine the problem is by running a set of controls with your experiment (see Tip 6 below).

In order for the ligation of your DNA insert and vector to work, it is critical to ensure that proper reaction conditions have been set. The ideal ligation reaction conditions are dependent on many factors, including the concentration of reaction components, reaction temperatures, and reaction times. If any of these factors are not optimized, the DNA ends of the insert and vector may fail to anneal frequently enough for ligase to seal the fragments together.

Here’s a recommended ligation reaction protocol that can serve as a starting point for your optimization:

* Ligation buffer includes ATP and DTT (a reducing agent), both of which degrade after multiple freeze-thaw cycles or extended incubations. In addition, DTT is prone to degradation during multiple exposures to oxygen, which also occurs through multiple freeze-thaw cycles. Since successful ligation is partly dependent on the correct concentrations of ATP and DTT, it is recommended to freeze ligation buffer in small single-use aliquots to prevent this freeze-thaw degradation.

** Blunt-end ligation is less efficient than sticky end ligation, so a higher concentration of ligase plus a crowding agent like polyethylene glycol (PEG) should be used for faster ligation.

Another optimization step is in the determination of the insert:vector ratio. The equation below can be used to calculate the even molar ratio in nanograms of insert DNA to vector DNA based on length:

To determine the best ratio of insert:vector to use for cloning, you may have to try different ratios ranging from 1:1 to 15:1, but a 3:1 ratio is a good place to start. For blunt-end ligation, be sure to adjust the insert:vector ratio and increase to 10:1 to optimize your result.

In general, T4 DNA ligase is a temperature-sensitive enzyme. Therefore, reaction efficiency and ligase activity decrease dramatically when the temperature is raised higher than 37°C. The ligation reaction should be incubated at room temperature. To increase reaction rates, the temperature can be cycled between the optimal temperature for the T4 DNA Ligase and the annealing temperature of the overhangs.

In general, prolonged incubation times are not necessary since ligase is a very efficient enzyme. Typically, the ligation should work at room temperature (~22°C) with reaction times ranging from ten minutes to one hour. In rare cases, such as when working with very long DNA fragments, overnight incubations of ligation reactions are necessary.

Several compounds can inhibit ligation reactions, including salts (like sodium chloride, potassium chloride, ammonium), EDTA, proteins, phenol, ethanol, and dATP. Since these inhibitors can be present in abundance, steps must be taken to either avoid concentrating the inhibitors or removing them from the reaction.

It can be tempting to concentrate the amount of vector and insert in your reaction by reducing the final volume (e.g., 10 µL versus 20 µL) of your ligation. However, while this will increase the concentration of DNA present, it may also increase the concentration of inhibitors present in the reaction. In general, a final ligation reaction volume of 20 μL is recommended since this volume includes ~10 μL of pure water (which is added to the reaction) to dilute any inhibitors that are present. In addition, by keeping your glycerol at less than 5% of the final reaction volume, this helps prevent excess glycogen from acting as an inhibitor in the ligase reaction.

Another option to deal with inhibitors present in your reaction is to remove them through a DNA purification stop. For this approach, commercially available silica-based columns are the preferred method of choice since the silica particles will not carry over through purification into the ligation reaction. If you choose to use silica matrix particles instead of columns, be aware that residual particles that may carry over can bind the ligase, thereby inhibiting the ligation reaction. If silica matrix particles are your only option, you will need to perform a short centrifugation step prior to the ligation reaction.

NOTE: If electrocompetent cells are being used for transformation following ligation, an additional column purification step of the ligated DNA should be used to remove any salt contaminants that may be present and therefore cause damage to your cells during electroporation.

When your cloning experiment didn’t work, it can be difficult to determine what went wrong. Since the success of your ligation reaction is crucial for cloning success, it is preferred to check your ligation reaction to confirm the insertion of your DNA insert into the vector before moving to the next step in your cloning workflow.

The confirmation of ligation reactions can be monitored using agarose gel electrophoresis. To do so, we recommend running the following samples out on a gel:

• DNA before ligation reaction (e.g., unligated vector plus DNA insert)
• DNA after ligation reaction (e.g., ligated vector plus DNA insert)

Before running your samples on the agarose gel, mix with an SDS-containing loading dye and incubate at 65°C for ten minutes. Adding the SDS to your loading dye allows for disassociation of ligase from the DNA in the sample, which prevents smearing on the gel. After the ten-minute incubation, run the samples on your gel. If your reaction was successful, the sample containing the ligated products would migrate at a higher molecular weight range than the sample with the unligated products.

Figure 3 illustrates how ligation reaction products may resolve on an agarose gel:

As shown in the gel, the ligation reaction appears:

Lane 1: The bright, clear bands represent the vector and DNA insert before ligation.

Lane 2: No SDS results in a smear in the lane as a result of the ligase that remains bound to the DNA.

Lane 3: With the addition of SDS, the ligase is no longer bound to the DNA, and clear higher molecular weight bands are seen, representing the ligation products in the reaction. In addition, the vector and DNA insert bands are diminished, indicated that the amount present has decreased.

Lane M: The size and approximate amount of ligation product, unligated vector and insert is determined by comparing bands to known size and amount of each band of a DNA ladder used as a marker.

Controls are critical components of any experiment. The table below lists common ligation reaction controls that can help track the success of the ligation steps in your cloning workflow.

Reaction setup	Purpose of the control and interpretation	Expected results
Uncut vector (No ligase)	✓ Checks quality and efficiency of competent cells ✓ Verifies antibiotic selection	Several colonies
Cut vector (No ligase)	✓ Determines background from uncut vector ✓ Checks restriction digestion efficiency	Few colonies
Cut vector + Phosphatase + Ligase	✓ Reveals background from any recircularized vector ✓ Checks efficiency of DNA phosphatase treatment	Few colonies
Insert or water + Ligase	✓Checks for contamination of reagents, stock    solutions, or pipettes	No colonies

As mentioned previously, T4 DNA ligase is a temperature-sensitive ligase and is inactivated at higher temperatures. If your ligase was stored or shipped improperly, it could be denatured or lose some activity. Since your ligation reaction can fail from due to denatured or impaired ligase, you should always follow the manufacturer’s recommendations for enzyme storage conditions and usage.

If you think your ligase may have been denatured or inactivated, check the following:

• The expiration date on the vial
  
• The ligase activity by using a positive control 

For example, the commercially available DNA Marker Lambda DNA/HindIII can be used as a positive control. When used in a ligation reaction with T4 DNA Ligase, the band pattern in the sample, when run on a gel, will show a single higher molecular weight band when the ligase is active. If the ligase is not active or is diminished, the original banding pattern of the Lambda DNA/HindIII marker will remain (multiple bands will show on the gel).

There are many reasons why ligation reactions can fail, with the most common arising from problems that occur before the addition of the T4 DNA ligase. When setting up or troubleshooting your ligation reactions, be sure to remember the tips listed below to help enable successful cloning results every time:

1. Consider your cloning strategy 
2. Check the ends of your DNA inserts 
3. Set up ideal reaction conditions 
4. Avoid inhibitors 
5. Visualize your ligation reactions on a gel 
6. Run controls 
7. Check to make sure your ligase is active 

To further improve your experiment, explore our extensive offering for ligation reactions in more detail:

• Thermo Scientific T4 DNA Ligase
• Rapid DNA Ligation Kit
• T4 polynucleotide kinase (T4PNK)
• FastAP Alkaline Phosphatase
• Fast DNA End Repair Kit

To learn more about different cloning strategies, read Common Cloning Applications and Strategies