Quick Tip - What is the ideal incubation temperature for ligation?

This video explains the best temperature at which to perform your ligation reaction.  Please note Hi-T4™ DNA Ligase is a thermotolerant ligase designed to function at higher temperatures than wild type T4 DNA Ligase. Hi-T4 DNA Ligase will function at temperatures as high as 50°C.

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What is the ideal incubation temperature for a ligation?

So, many people use a 16°C overnight ligation. However, we find that in most cases actually room temperature for a shorter incubation time works really well.  In fact, if you want to use some of our more advanced ligation formulations, such as the Quick Ligation Kit or the Blunt/TA or Instant Sticky-end master mixes, room temperature incubation is specifically recommended.

An important side note: if you’re using NEB’s formulations, never heat inactivate your ligation reaction. Heat inactivation in these cases can lead to loss of DNA and a reduced transformation efficiency.

And remember, if you have any additional questions, you can always contact us via email at [email protected]

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