T4 DNA Ligase catalyzes the
formation of a phosphodiester linkage between 5'-phosphoryl group and
adjacent 3'-hydroxyl group of duplex DNA or RNA in a blunt-ended or
cohesive-ended configuration. ATP is required for the reaction.
Recombinant strain of E. coli.
¡Ü Joining of
double-stranded DNA fragments with cohesive ends or blunt ends.
One Weiss unit is
defined as the amount of enzyme required to catalyze the exchange of
1nmole of 32P from pyrophosphate to ATP, into Norit-adsorbable material in
20 minutes at 37¡É. The reaction conditions for Weiss unit definition assay
are : 66mM Tris-HCl (pH 7.6), 6mM MgCl2, 1mM DTT, 0.066mM ATP and 3.3¥ìM
[32PPi]. One Weiss unit is equivalent to 67 cohesive end ligation unit
which is the amount of enzyme required to give 50% ligation of Lambda DNA
Hind III fragments in 30 minutes at 16¡É in 20¥ìl reaction mixture and a 5'
DNA termini concentration of 0.12¥ìM (300¥ìg/ml).
STANDARD UNIT ASSAY CONDITIONS
Tris-HCl (pH7.8), 10mM MgCl2, 10mM DTT, 1mM ATP.
CONCENTRATION: 2-4 units/¥ìl, 10-20 units/¥ìl
(High concentration product)
10X REACTION BUFFER
(pH7.8), 100mM MgCl2, 100mM DTT, 10mM ATP.
10mM Tris-HCl (pH
7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. Store
1. Helfman, D.M. et al.
(1987) Methods Enzymol. 152, 349. 2. Weiss, B. et al. (1968) J. Biol.
Chem. 243, 4543.