Product Description
T4 DNA ligase catalyzes the formation of a phosphodiester bond between 5’ phosphate and 3’ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

Applications

Cloning of restriction fragments
Joining linkers and adapters to blunt ended DNA

Source of Protein
A recombinant E. coli strain carrying the cloned T4 DNA Ligase gene

Components
1. T4 DNA Ligase supplied in 10 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 50% Glycerol.

2. 10X T4 DNA Ligase Reaction Buffer containing ATP.

Storage Conditions
Store at –20°C.

Concentration
400 units/µL (regular format)
2,000 units//µL (high concentration format)

Unit Definition
One Weiss Unit is approximately equivalent to 67 Cohesive End Ligation Units

Quality Assurance
Purified free of contaminating endonucleases and exonucleases. In addition, enzyme purity is analyzed by SDS-PAGE, and negligible E.coli genomic DNA is confirmed by qPCR.

Suggested Reaction Conditions
1X T4 DNA Ligase Reaction Buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol,1 mM ATP, 25 µg/mL bovine serum albumin. Recommended DNA concentration (0 .1 to 1 µM in 5´ termini). Optimal ligation occurs at 16°C.

Notes on DNA Concentration
The overall concentration of vector + insert should be between 1-10 μg/mL. In general, a 3:1 molar excess of insert is recommended when cloning a fragment into a plasmid vector. Insert:vector ratios below 2:1 result in lower ligation efficiency. Insert:vector ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations using different insert:vector ratios.

Suggested Protocol
For convenience, ligations may be done at room temperature (20–25°C).

For cohesive (sticky) ends, use 1 µL of T4 DNA Ligase in a 20 µL reaction for 10 minutes
For blunt ends, use 1 µL of T4 DNA Ligase in a 20 µL reaction for 2 hours or 1 µL high concentration T4 DNA Ligase for 10 minutes

Alternatively, Marligen's Rapid Ligation Kit (Cat. No, 11620-030 (30 reactions) or Cat. No. 11620-150 (150 reactions) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

Important Note: The T4 DNA Ligase Reaction Buffer contains ATP, which is an essential cofactor for the reaction. Therefore, we recommend thawing T4 DNA Ligase Reaction Buffer on the bench or in the palm of your hand and not at 37°C (this prevents breakdown of ATP). Once thawed, the buffer should be placed on ice. BSA (250 µg/mL) in the Ligase Reaction Buffer may precipitate when thawed. This will not affect ligation efficiency.