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  • FAQ: Which ligase should I use?

    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.