T4 DNA Ligase

Source: E. coli strain containing an overproducing clone of T4 DNA Ligase.

Applications:

1. Ligation of cohesive-end or blunt-end termini.
2. Ligation of synthetic linkers or adaptors.
   

Description:
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed
5' phosphoryl and 3'-hydroxyl termini in duplex DNA or RNA. It repairs single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids and will join both blunt-end and cohesive-end fragments of duplex DNA or RNA⁽¹⁾.

Properties:
Molecular Weight: 55 kDa
Optimum pH: 7.5 - 7.8
Optimum Temperature: 16°C for exchange reaction
Requirements for Divalent Cation: Mg²⁺, Mn²⁺
Optimum Mg²⁺ Concentration: 10mM
Sulfhydryl Requirement: 2-mercaptoethanol, dithiothreitol
Stimulators: Spermine (0.5 to 1mM), Spermidine (0.5 to 1mM)
Inhibitors: Na (>0.2M), K (>0.2M)
Inactivation: Heat at 65°C for 10 minutes

Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, exonucleases and ribonucleases.

Storage Buffer:
25mM Tris-HCl (pH 7.6), 100mM NaCl, 1mM DTT, 0.5mM EDTA, 50% glycerol.

Assay Conditions:
T4 DNA Ligase is assayed according to the pyrophosphate exchange method of Weiss
et al. The reaction mixture (300 µl) contains 67mM Tris-HCl, (pH 7.8), 6.7mM MgCl₂, 10mM DTT, 66µM ATP and 3.3µM radiolabeled pyrophosphate. Incubation is at 37°C for
20 min.

Note: ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA Ligase which requires NAD.

Unit Definition:
One Weiss unit is the amount of enzyme required to convert 1 nmol of radiolabeled phosphate from pyrophosphate into absorbable material in 20 min at 37°C under standard assay conditions. One Weiss unit equals about 67 cohesive end ligation units.

Concentration:
PN 70005: 1 unit/µl; PN 70042: 10 units/µl

Tested User Friendly^TM Functional Test:
Re-ligation of linearized plasmid DNA > 80% of control, as determined by counting transformed bacterial colonies.

Functionally Tested 10X T4 DNA Ligase Reaction Buffer (1 ml included, PN 70087): 660mM Tris-HCl (pH 7.6), 66mM MgCl₂, 100mM DTT, 660µM ATP

T4 DNA Ligase Dilution Buffer (1 ml included, PN 70108): 20mM Tris-HCl (pH 7.6), 60mM KCl, 5mM DTT, 1mM EDTA, 50% glycerol

References:

1. ROSSI, R., MONTECUCCO, A., CIARROCHI, G. AND BIAMONTI, G. (1997) Nucl. Acids Res. 25 (11), 2106-2113.
2. WEISS, B., JACQUEMIN-SABLON, A., LIVE, T. R., FAREED, G. C. AND RICHARDSON, C. C. (1968) J. Biol. Chem. 243, 4543-4555.

Storage:
Shipped on dry ice. Store at -20°C.