FAQ: When should Hi-T4 DNA Ligase be the enzyme of choice?

Whereas T4 DNA Ligase is steadily inactivated at temperatures >37°C, Hi-T4 DNA Ligase can withstand temperatures as high as 50°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), or requires multiple high-temperature/low temperature thermal cycles (i.e., Golden Gate Assembly protocols).