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5 DNA Ligation Tips

DNA Ligation Tips

DNA ligations can be frustrating. Sometimes they don’t work for no obvious reason. Our top 5 DNA ligation tips should improve the efficiency of your ligations, and will hopefully increase your cloning success rate!

1. Aliquot the ligase buffer

The ATP in the ligase buffer is essential for the DNA ligation reaction, but is broken down by repeated freeze-thaw cycles. To avoid this, aliquot the ligase buffer from each new stock of DNA ligase. Make the aliquots small enough for single-use (e.g. 5 µL), and make sure to completely defrost and mix your buffer well before you aliquot.

2. Heat the DNA just before ligation

When setting up a cohesive-ended ligation, mix the vector and insert fragments first and heat to 65°C for 5 minutes before adding the remaining reaction components. This heating step disrupts any vector/vector or insert/insert cohesive-end interactions that may otherwise interfere with the desired vector/insert interaction, reducing ligation efficiency.

3. Check the pH

The optimum pH range for DNA ligation is between 7.6 and 8.0. Depending on how the DNA fragments were prepared, the pH of your ligation mixture may lie outside of this range. You can check the pH of your ligation mixture by pipetting approximately 0.2 µL of the mix onto narrow range pH paper (e.g. pH 6-8). If required, adjust the pH using 0.2 µL drops of 2M Tris base or 1M HCl.

4. Include polyethylene glycol (PEG)

As with any chemical reaction, the concentration of the reaction components can greatly influence the speed of the ligation reaction. PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. Adding PEG (e.g. PEG 8000) to a final concentration of 5-15% may increase ligation efficiency. Bear in mind however that PEG concentrations above 5% can reduce transformation efficiency. In addition, heat inactivation or extended incubation of ligation reactions containing PEG can also decrease transformation efficiency.

5. Add a restriction enzyme just before transformation

This neat trick can be used to circumvent high background resulting from undigested vector. If the vector fragment removed during the preparative digest contains a unique restriction site, adding the respective restriction enzyme to the ligation reaction will selectively digest any intact vector, preventing it from being transformed. Adding 1 µL of the enzyme 5-10 minutes prior to transformation should be sufficient.

Originally published on August 24, 2007.  Revised and updated on June 1, 2016.

Image Credit: Colin Davis


  1. Giulia on November 28, 2016 at 9:02 am

    Hi Nick,
    interesting article!
    What do you think about dephosphorylating the plasmid before ligation with alkaline phosphatase (CIP)? Will this give you an easier ligation?

  2. Anil Raj on September 9, 2016 at 12:23 pm

    Dear Nick,
    As I went on to reading the post, I read “PEG is a hydrophobic molecule”, which I believe is wrong. PEG is hydrophilic molecule which by means of solvent exclusion effect causes the water soluble ingredients to get condensed in the little water available to them.

  3. Anil Raj on September 9, 2016 at 10:54 am

    Dear Nick,
    Greetings from India, thanks a lot for maintaining this blog. About this heating DNA (vector plus insert) mix before adding ligase may, as you said, increase ligation efficiency. But the proposed reason behind it, I’m not getting it. Because in the mix, there will be vector-vector , insert-insert additions including vector-insert additions as well, which when we heat can be broken again. Also, if we are to cool it before adding ligase, the same thing will happen again.

    • Dharmesh on April 29, 2017 at 4:29 pm


      We don’t store them in the same tube (vector and insert). I think he is more talking about intra-interactions, not inter-interactions. Intra-interactions may take place during storage.

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