FAQ: I observed high backgrounds with empty vector when using NEBridge Ligase Master Mix. What should I do?

We suggest performing the transformation immediately after the reaction is done. If you store the reactions at -20°C overnight or for an extensive period, you will need to do another 60°C incubation for 5 minutes right before the transformation. 60°C favors the restriction enzyme activity over T4 DNA Ligase  activity and can reduce background by digesting any “re-ligated” vector that still carries the restriction enzyme site.