T4 DNA Ligase

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids, joins DNA fragments with either cohesive or blunt termini. The T4 DNA Ligase requires ATP as a cofactor.

The enzyme is available in 250 and 1,250 unit sizes at a concentration of 5 U/µL. The enzyme is supplied with a 10× T4 Ligation Buffer.

Applications

• Routine subcloning.
• Recircularization of linear DNA.
• Library construction.
• Linker ligation.

Product Specifications

• Storage Buffer: 10 mM Tris-HCl (pH 7.4 at 25 ^oC), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 50% (v/v) glycerol.
• 10× T4 Ligation Buffer: 500 mM Tris-HCl (pH 7.4 at 25 ^oC), 100 mM MgCl_2, 10 mM ATP, 100 mM DTT.
• Unit Definition: One Weiss unit of the enzyme catalyzes the conversion of 1 nmol of [³²PPi] into Norit-adsorbable form in 20 min at 37°C.
• Enzyme Activity Assay Conditions: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 0.066 mM ATP, 10 mM DTT, 3.3 μM [³²PPi].
• Source: A recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.

General Protocol

1.   In a microcentrifuge tube, combine the following reagents for a 20 µL ligation reaction:

      10× T4 Ligation Buffer              2 µL
       Vector DNA                             10-100 ng
      Insert DNA                               As required*
      T4 DNA Ligase                        1 U or 5 U**
      Nuclease-free water                  up to 20 µL

     Note: * For cohesive-end ligations, use a 1:1 or a 3:1 molar ratio of insert:vector; for blunt-end ligations, use a 3:1 molar ratio of insert:vector DNA.
              ** For cohesive-end ligations, use 0.2 µL (1 U) T4 DNA Ligase; for blunt-end ligations, use 1 µL (5 U) T4 DNA Ligase.

2.  Mix gently and spin down for a few seconds.

3.  Incubate at room temperature for 1 hour.

4.  Immediately transform competent cells with 2 μL of the ligation reaction.