New England Biolabs, M0205L, E. coli DNA Ligase

DNA Ligase will ligate these substrates: Related Categories DNA Ligases Applications Non-Cloning Ligation Specification Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5' DNA termini concentration of 0.12 μM, 300 μg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X E. coli DNA Ligase Reaction Buffer. Reaction Conditions 1X E. coli DNA Ligase Reaction Buffer Incubate at 16°C 1X E. coli DNA Ligase Reaction Buffer 30 mM Tris-HCl 4 mM MgCl2 26 µM NAD 1 mM DTT 50 µg/ml Recombinant Albumin (pH 8 @ 25°C) Usage Concentration 10,000 units/ml Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: How does E. coli DNA Ligase (NEB# M0205) differ from T4 DNA Ligase (NEB# M0202)? A: Under recommended conditions E. coli DNA Ligase will not join blunt ends nor DNA to RNA. E. coli DNA Ligase is more specific for cohesive ends than T4 DNA Ligase. Q: Which ligase should I use? A: The Quick Ligation Kit (NEB #M2200) should be used if time is a factor especially if the ligation involves blunt ends ( 5 mins RT). T4 DNA Ligase (NEB #M0202) Is the enzyme of choice for the majority of recombinant DNA applications. It can be used for for cohesive ends (10 mins RT) and blunt ends (2 hours RT) or if the ligation is to be done overnight. Transformation efficiency can drop if the Quick Ligation Kit is used overnight. Use T4 DNA Ligase if heat inactivation is required. A spin column is required for the removal of PEG from the Quick Ligation Kit prior to electroporation. Concentrated ligase is suggested for ligating single base overhangs. Large constructs such as BACs (bacterial artificial chromosomes) which require longer incubation times to circularize benefit from high concentration ligase. Use E. coli DNA Ligase (NEB #M0205) if cohesive end ligation is desired while suppressing blunt end ligation or ligation to RNA. E.coli DNA Ligase is more specific for cohesive ends than T4 DNA Ligase. Use T4 RNA ligase (NEB# M0204) if the goal is to ligate single stranded DNA or RNA. Q: Will E. coli DNA Ligase work in rCutSmart Buffer? A: E. coli DNA ligase will work in rCutSmart Buffer, with the addition of NAD. To see its % functional activity in rCutsmart, and that of other DNA modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.