When is a long incubation time necessary?
For some substrates such as single base overhangs or blunt ends, as well as for very large DNA fragments greater than 5 kb, are more difficult to ligate than typical short fragments with cohesive ends. For these substrates, you’ll need a longer incubation time for efficient ligation.
You can find, in the protocol for ligation with T4 DNA ligase at NEB.com, recommended incubation times for different substrates. As an alternative to long incubation times, you can also use products specifically formulated for difficult ligations, including the Blunt/TA Master Mix and the Quick Ligation Kit.
Remember, if you have any additional questions, you can always contact us via email at firstname.lastname@example.org.
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