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 dH2O	Up to 19ul
T4 DNA Ligase***	1ul
Total Volume	20ul

*: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
NP100052	T4 DNA Ligase 10X T4 DNA Ligase Reaction Buffer	400,000 U/mL	16,000U
NP100051	T4 DNA Ligase (High Concentration) 10X T4 DNA Ligase Reaction Buffer	2,000,000 U/mL	80,000U