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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.

  4. Tere on February 23, 2016 at 6:44 pm

    Hi Nick!
    Which ratio vector/insert do you sugest for 1 kb inserts? Would you recomend some internet ratio calculator?

  5. boffin on October 21, 2011 at 7:31 pm

    hey nick ,,, i find that most of your tech tips (both from you and the blog) are applicable and helping a lot to fine tune my experiments and results !!!

    thanks and good luck !!
    And now, its my anxiety to ask you that I have read from few company manuals that PEG strictly inhibits ligase and so to avoid that !?? do you have any technical/ logic for using PEG during ligase reaction.

  6. Pete on March 24, 2010 at 12:33 pm

    Hi, great article! Just a quick question about PEG, though… I don’t really understand the rationale behind this. If it increases the concentration by just taking up space, then why not just run the remaining ingredients at that higher concentration in less volume (e.g. don’t add water)?

  7. LXu on April 23, 2009 at 4:33 am

    Hi Nick,
    I really appreciate the articles you’ve been posting. I’m wondering about the heating the DNA prior to ligation step. T4 DNA ligase is inactivated by heating it at 65 degrees C for 10 min. I know you are heating the DNA only at this step, but do you allow it to cool before adding the ligase? My only worry is that if the DNA is still around 60-65 degrees when the ligase is added, it’ll lower its efficiency…


    • Nick on May 12, 2009 at 7:51 am

      Hi LXu.

      Yes, the DNA solution should be allowed to cool before adding the ligase. However the volume of the ligation mix is so small that it should cool down very quickly.

  8. che on April 21, 2009 at 6:26 am

    Hi Nick,
    Thanks alot for this informative forum.
    Do you have any suggestions on how to improve the ligation efficiency of blunt ened products?

  9. M. Dadashi on April 7, 2008 at 8:16 am

    Dear Nick!

    Thank you very much for your nice comments.
    You have written good comments on vector preparation and ligation. But I could not find, which protocol you are using for vector preparation !

    Actually, I have problem with my transformation after ligations. Usually I get a few ( and sometimes no) colonies. Now I am working with a pET28b vector and i just wanna subclone a gene from pUC19 into this vector. After a few months of hardworking, I got only one of my mutants but I still have problem to get other mutants in pET28b.

    I need these mutants for rapid purification of them by Ni-NTA column.

    It would be very appreciated if you help me to solve this problem forever!

    By the way, I use SAP for vector preparation and following that i pass phenol- chloroform, Diethylether, Treatment with NaCl- PEG , Precipitation with NaAcetate plus 99% Ethanol and washing with 70% Ethanol.

    For gene preparation, I digest the gene following amplification, and after that I usually use a short time UV translumination for separation of the gel containing my gene and after that I have QIAGEN kit extraction of the gene.

    With my best wishes,

    Mohammad DADASHIPOUR,
    [email protected]
    [email protected]

  10. Nick on December 14, 2007 at 9:00 pm

    Hi Marc,

    Right now I use a 30 min incubation at room temperature, but I use a rapid ligation kit.

    The problem could be with the vector prep – are you using EtBr/UV to visualise the digested vector? This can really reduce cloning efficiency. See: https://bitesizebio.com/2007/08/22/cloning-tips-vector-prep/

    Otherwise, drop me an email (using the contact button above) if you want to discuss it further.

  11. Marc on December 14, 2007 at 6:26 pm

    Hi Nick,

    I’m curious… how long and at which temperature do you incubate your ligation reaction? What works best for you?
    I’m right now careful and do ON @ 4 °C for sticky ends… but I still get only few clones 🙁

    Great blog by the way… I just discovered it.


  12. Nick on September 18, 2007 at 8:04 am

    Hi Aitor

    As far as I am aware, there is no literature on this – I believe it has been empirically observed that the heating step improves ligation, and that the most likely explanation is that the heating disrupts vector/vector or insert/insert interactions.

    I certainly see an improvement when I use this step and the proposed mechanism makes sense to me!

  13. Aitor on September 17, 2007 at 10:57 am

    Very usefull, tahnk you! Could you tell me where I can find more information (papers) on heating DNA before ligation? Thanks

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