New England Biolabs, M2622S, Hi-T4™ DNA Ligase

Looking for additional T4 DNA Ligase formulations and variants? Related Categories DNA Ligases Applications BioBrick, ®, Assembly,, Cloning Ligation Specification Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of 6 µg of Lambda-HindIII DNA in 30 minutes at 25°C in a total reaction volume of 20 μl. Reaction Conditions 1X T4 DNA Ligase Reaction Buffer Incubate at 25°C 1X T4 DNA Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 10 minutes FAQ Q: What are some causes of ligation reaction failure that can lead to transformation failure? A: Ligation failed because there was no ATP or Mg2+. Use the supplied buffer or add ATP to a compatible buffer. ATP in buffers older than one year may have degraded enough to cause problems. When supplementing with ATP, be sure to use riboATP as deoxyriboATP will not work. Ligation failed due to EDTA in the reaction. Clean up the DNA (we recommend Monarch® nucleic acid purification kits). CIP, BAP, Antarctic Phosphatase or SAP are not completely inactivated following dephosphorylation step. Follow the recommended procedure to remove the phosphatase. Concentration was too high resulting in ligation only producing linear DNA. Keep the total DNA concentration between 1-10 μg/ml. The insert and the plasmid do not have phosphates. Note: primers may not have phosphates, preventing blunt-end ligation with vectors that have been treated with CIP. Order primers with phosphates or phosphorylate the primers with T4 Polynucleotide Kinase prior to use. Too much ligation mixture was added to the cells. Add between 1-5 μl to 50 μl competent cells. The insert was large and the conditions didn't allow circularization. Reduce the insert concentration. The ligase was inactive. Test on Lambda HindIII DNA or other convenient substrate. The ligation mix contained PEG and was incubated overnight. Extended ligation with PEG causes a drop off in transformation efficiency. This could be due to the gradual production of large linear pieces of DNA that can inhibit transformation. StickTogether™ DNA Ligase Buffer contains PEG. The ligation mix was not purified prior to electroporation. The buffer must be removed or a spark will be generated by the salt. Dialyze the sample or use a spin column to purify (we recommend Monarch nucleic acid purification kits). The PEG in StickTogether™ DNA Ligase Buffer prevents sparking but it also prevents electroporation. PEG must be removed using a spin column. Q: When should Hi-T4 DNA Ligase be the enzyme of choice? A: Whereas T4 DNA Ligase is steadily inactivated at temperatures >37°C, Hi-T4 DNA Ligase can withstand temperatures as high as 45°C for extended periods (up to 72 hours) without any significant loss in activity. Therefore, Hi-T4 DNA Ligase should be the enzyme of choice if the ligation reaction is to be carried out at a higher temperature (>37°C) for an extended length of time (> 15 minutes). Q: Which buffer should I use with Hi-T4 DNA Ligase? A: If trying to ligate cohesive ends, we recommend using Hi-T4 DNA Ligase in 1X T4 DNA Ligase Buffer. If trying to ligate blunt ends or single-base overhangs, Hi-T4 DNA Ligase should be used in our PEG-containing StickTogether™ DNA Ligase Buffer. Do not heat kill the reaction, as heat treating the PEG in the StickTogether™ DNA Ligase Buffer will inhibit transformation. Q: What is the activity of Hi-T4 DNA Ligase in other buffers? A: Hi-T4 DNA Ligase is 100% active in T4 DNA Ligase Buffer. Like T4 DNA Ligase, Hi-T4 DNA Ligase has reduced activity in NEBuffer 3/r3.1 and HiFi Taq DNA Ligase Buffer due to their high salt content. Buffer % Activity1 T4 DNA Ligase Buffer 100% NEBuffer r1.12 100% NEBuffer r2.12 100% NEBuffer r3.12 50% rCutSmart® Buffer2 100% Taq DNA Ligase Buffer2 100% HiFi Taq DNA Ligase Buffer2 50% 1Activity relative to activity on a cohesive end substrate in T4 DNA Ligase Buffer (25°C for 10 minutes). 2 Supplemented with 1 mM ATP. Please be sure to use riboATP (NEB #P0756) as deoxyriboATP will not work. To see its % functional activity in CutSmart, and that of other DNA modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in CutSmart® Buffer chart. Q: Can this ligase be heat inactivated? A: Yes, just like T4 DNA Ligase, this ligase can be heat inactivated for 10 minutes at 65°C. Do not heat inactivate if there is PEG in the reaction buffer (StickTogether™ DNA Ligase Buffer), as transformation will be inhibited. Q: What is the difference between the unit definitions of T4 DNA Ligase (NEB #M0202) and Hi-T4 DNA Ligase (NEB #M2622)? A: Both T4 DNA Ligase and Hi-T4 DNA Ligase unit definitions are defined as 50% ligation of 6 μg HindIII fragments, however, the incubation temperature differs. The T4 DNA Ligase unit definition is determined at 16°C, while the Hi-T4 DNA Ligase unit is determined at 25°C. Q: How much DNA should be used in a ligation using this ligase? A: The unit definition uses 6 μg HindIII fragment. This high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Vector:Insert molar ratios between 1:1 and 1:10 are recommended (1:3 is typical). If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.