T4 DNA Ligase

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme joins blunt end and cohesive end termini as well as repairs single-stranded nicks in duplex DNA and some DNA/RNA hybrids. T4 DNA Ligase is applicable to cloning restriction fragments and to joining linkers and adapters to blunt-ended DNA.

T4 DNA Ligase Protocol

  • Set up the following reaction (based on the composition table) in a microcentrifuge tube on ice.
  • Short centrifugation after gentle percussion.
  • Gently mix the reaction by pipetting up and down and microfuge briefly.
  • For cohesive (sticky) ends, incubate at 16℃ overnight or room temperature for 10 minutes.
  • For blunt-ends or single-base overhangs, incubate at 16℃ overnight or room temperature for 2 hours (alternatively, high-concentration T4 DNA Ligase can be used in a 10-minute ligation).
  • Heat inactivate at 65℃ for 10 minutes.
  • Chill on ice and transform 1-5ul of the reaction into 50ul competent cells.
Composition Amount
10X T4 DNA Ligase Reaction Buffer*2ul
Vector DNA (4kb)50ng (0.02 pmol)
Insert DNA (1kb)**37.5ng (0.06 pmol)
Nuclease-free dH2OUp to 19ul
T4 DNA Ligase***1ul
Total Volume20ul

*:10X T4 DNA Ligase Reaction Buffer should be thawed and resuspended at room temperature.
**:Insert DNA (1 kb): A ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
***:T4 DNA Ligase should be added last.

T4 DNA Ligase

Catalog # Components Concentration Size
  • T4 DNA Ligase
  • 10X T4 DNA Ligase Reaction Buffer
400,000 U/mL16,000U
  • T4 DNA Ligase (High Concentration)
  • 10X T4 DNA Ligase Reaction Buffer
2,000,000 U/mL80,000U