DNA ligation is a commonly used method in molecular cloning to physically join vector DNA to a gene of interest.
It is a relative straightforward technique which involves mixing of DNA insert and vector DNA together with a DNA ligase followed by incubation, heat inactivation, and final transformation. Still, ligation sometimes fails leaving us with time-consuming troubleshooting and laborious additional work. Potential problems causing ligation failure can be to high salt or EDTA in the reaction, no ATP or Mg2+ or temperature alterations during the ligation process.
Here we present two new T4-DNA Ligase variants that have been tailored to deal with either high-salt or high temperatures!
Saving Time and Maximizing Ligation Efficiency at High-Salt Concentration
Salt, a carry-over from reaction components such as the vector or insert DNA, decreases the ligation efficiency of T4 DNA Ligase. Thus, an additional step of ethanol precipitation is necessary to clean up the DNA before the actual ligation process. So, if you want to save time by not cleaning up your DNA why not try out NEBs new salt-tolerant variant of T4 DNA ligase. The Salt-T4 DNA Ligase (NEB#M0202) functions at high salt concentrations up to 500 mM for the ligation of cohesive ends and up to 150 mM for the ligation of blunt ends (Fig.).
Maximizing cloning efficiency by temperature-cycle ligation
Are you belong to a group of designers that like to create DNA constructs consisting of multiple DNA fragments? The Golden Gate Assembly assay (NEB#E1601) from NEB allows cloning of several restriction fragments at once. Following the protocol that involves several temperature-shift cycles a thermostable ligase should be the enzyme of choice. Thus, NEB has developed a thermotolerant Hi-T4 DNA Ligase (NEB#M2622) that functions at elevated temperatures as high as 45oC without any significant loss in activity (Fig.).
So why not try out the Hi-T4 DNA Ligase for your next ligation requiring higher temperature for an extended length of time!