Home Technical Resource > DNA/RNA Modifying Enzymes & Cloning Technologies >
Maximizing Ligation Efficiencies
NEB T4 DNA Ligase is the most extensively used ligase for cloning-based experiments. Typically, a ligation reaction (blunt or cohesive ends) using traditional T4 DNA Ligase (NEB #M0202) 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 #M0202T). Alternatively, NEB’s Quick Ligation Kit (NEB #M2200) is uniquely formulated to ligate blunt or cohesive ends in 5 minutes at room temperature. The following tips will help to achieve maximum results from your ligation reactions.
Reaction Buffers
- T4 DNA Ligase Buffer 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.
- BSA in the Ligase Buffer may precipitate when thawed. This will not affect ligation efficiency.
- Ligations can be performed in any of the four standard restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP
- When supplementing with ATP, use ribo ATP. Deoxyribo ATP will not work.
- Before ligation completely inactivate restriction enzyme by heat inactivation, spin column or Phenol/EtOH purification.
DNA
- Use purified DNA preparations without EDTA or high salt concentrations.
- Either heat inactivate (Antarctic Phosphatase) or remove phosphatase (CIP, BAP or SAP) before ligation.
- Keep total DNA concentration between 1-10 µg/ml.
- Insert:Vector molar ratios between 2:1 and 6:1 are optimal for single insertions.
- Insert:Vector molar ratio should be 6:1 to promote multiple inserts.
- If you are unsure of your DNA concentration, perform multiple ligations with varying ratios.
Ligase
- For most ligations (blunt or cohesive) traditional T4 DNA Ligase or the Quick Ligation Kit are recommended.
- For single base overhangs, it is recommended to use up to 5 µl concentrated ligase at 16°C overnight.
- For large inserts, reduce insert concentration and use concentrated ligase at 16°C overnight.
- T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes.
- Do not heat inactivate if there is PEG in the reaction buffer because transformation will be inhibited. The Quick Ligation Kit contains PEG.
Transformation
- 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 to purify first.