E. coli DNA Ligase

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

Applications:

1. Ligation for cloning^(1,3).

Description:
E. coli DNA Ligase, in the presence of NAD, catalyzes the formation of the phosphodiester bond in duplex DNA containing cohesive ends (juxtaposed 5'-phosphoryl and 3'-hydroxyl termini). Also, in the presence of certain macromolecules such as PEG, E. coli DNA Ligase catalyzes blunt end ligation of DNA⁽¹⁾. The enzyme is purified by the procedure of Panasenko, et al.⁽²⁾. E. coli DNA Ligase has been employed in a procedure for high efficiency cloning of full-length cDNA⁽³⁾.

Properties:
Molecular Weight: 75 kDa
Optimum pH: 8.1
Optimum Divalent Cation Concentration:
    Mg²⁺: 3mM
    Mn²⁺: 6mM

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

Storage Buffer:
10mM Tris-HCl (pH 7.5), 50mM KCl, 0.1mM EDTA, 10mM ammonium sulfate, 1mM DTT, 50% glycerol.

Assay Conditions:
The reaction mixture (20 µl) contains 30mM Tris-HCl (pH 8.0), 4mM MgCl₂, 1.2mM EDTA, 1mM DTT, 26µM NAD, 50 µg/ml BSA and Hind III fragments. Incubation is at 16°C for
30 min.

Unit Definition:
One unit is the amount of enzyme required to give 50% ligation of Hind III fragments in
30 min at 16°C with a 5'-DNA termini concentration of 0.003µM (7 µg/ml) under standard assay conditions.

Concentration:
10 units/µl

Tested User Friendly^TM Functional Test:
Ligation of Hind III fragments. One unit gives ~50% ligation in 30 min at 16°C. Verified by agarose gel electrophoresis.

PROTOCOL FOR LIGATION USING E. coli DNA LIGASE
For a 50 µl Reaction:
    5 µl 10X Ligation Buffer
    1 µg DNA
    0.1mM NAD
    10 units E. coli DNA Ligase

Incubate at 10°C to 25°C for 2 to 16 hrs. Stop reaction by adding 2 µl of 0.5M EDTA or by heating to 75°C for 10 min.

Functionally Tested 10X E. coli DNA Ligase Reaction Buffer (1 ml included,
PN 70098):
300mM Tris-HCl (pH 8.0), 40mM MgCl₂, 260 µM NAD, 10mM DTT, 0.5 mg/ml BSA.

References:

1. ZIMMERMAN, S. B. AND PHEIFFER, B. H. (1983) Proc. Natl. Acad. Sci., USA 80, 5852-5856.
   
2. PANASENKO, S. M., ALAZARD, R. J. AND LEHMAN, I. R. (1978) J. Biol. Chem. 253, 4590-4592.
   
3. OKAYAMA, H. AND BERG, P. (1982) Mol. Cell. Biol. 2, 161-170.
   
4. PANASENKO, S. M., CAMERON, J. R., DAVIS, R. W. AND LEHMAN, I. R. (1977) Science 196, 188-189.
   

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