Tips for Maximizing Ligation Efficiencies

NEB T4 DNA Ligase (NEB #M0202) is the most extensively used ligase for cloning-based experiments. Traditionally, a ligation reaction (blunt or cohesive ends) using traditional T4 DNA Ligase involves incubation at 16°C using 0.1-1 µM DNA (5´ termini) in 1X T4 DNA Ligase Buffer. For your convenience, T4 DNA Ligase can also be used at room temperature, and is available in concentrated form (NEB #M0202). Alternatively, NEB’s Quick Ligation™ Kit (NEB #M2200) is uniquely formulated to ligate blunt or cohesive ends in 5 minutes at room temperature. More recently, new formulations of T4 DNA Ligase have been commercialized for fast and efficient ligation of all types of DNA ends. DNA Ligase Master Mixes are pre-mixed, ready-to-use formulations that are specially formulated for Blunt/TA or Sticky Ends. The tool NEBCloner can help you select the best ligase formulation for your needs.

The following tips will help to achieve maximum results from your ligation reactions.

Reaction Buffers

  • T4 DNA Ligase Buffer (NEB #B0202) should be thawed on the bench or in the palm of your hand, and not at 37°C (to prevent breakdown of ATP).
  • Once thawed, T4 DNA Ligase Buffer should be placed on ice.
  • Ligations can be performed in any of the four standard restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer (NEB #B0201) if they are supplemented with 1 mM ATP.
  • When supplementing with ATP, use ribo ATP (NEB #P0756). Deoxyribo ATP will not work.
  • Before ligation, completely inactivate restriction enzyme by heat inactivation, spin column (NEB #T1030), or Phenol/EtOH purification.


  • Heat inactivate (Antarctic Phosphatase, Quick CiP, rSAP) before ligation.
  • Keep total DNA concentration between 1-10 µg/ml.
  • Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Insert: vector molar ratio should be 6:1 to promote multiple inserts. Use NEBioCalculator to calculate molar ratios.
  • For cloning more than one insert, we recommend NEBuilder® HiFi DNA Assembly Products.
  • If you are unsure of your DNA concentration, perform multiple ligations with varying ratios.


  • For most cohesive-end ligations, standard T4 DNA Ligase, Instant Sticky-End Ligase Master Mix, or the Quick Ligation Kit are recommended.
  • For blunt and single-base overhangs, Blunt/TA Ligase Master Mix is recommended.
  • For ligations that are compatible with electroporation, ElectroLigase (NEB #M0369) is recommended.
  • Standard T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes.
  • Do not heat inactivate the Quick Ligation Kit or ligase master mixes.


  • Add between 1-5 µl of ligation mixture to competent cells for transformation.
  • Extended ligation with PEG causes a drop off in transformation efficiency (Quick Ligation Kit).
  • Electroporation is recommended for large constructs (>10,000 bp). Dialyze sample or use a spin column (NEB #T1030) to purify first if you have used the Quick Ligation Kit or ligase master mixes.
  • For ligations that are compatible with electroporation, ElectroLigase is recommended.


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