This enzyme is supplied in a buffer of 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM 2-mercaptoethanol, 0.1 mM EDTA and 50% glycerol. It was purified from an E. coli strain expressing a recombinant clone of T4 DNA ligase. Its molecular Weight is 68kDa.
Requirements: Mg2+, ATP and DTT. The optimum concentration of Mg2+ is 10mM. Mn2+ may be
substituted for Mg2+ but is only 25% as effective as Mg2+.
Inhibition: 50% inhibition by greater than 150mM NaCl (activity measured at nicks. Other inhibitors
include 0.2M K+, Cs+, Li+, NH4+ and 1mM spermine.
Inactivation: Heat to 70°C for 10 minutes.
Contents: GB-Clone™ T4 DNA Ligase (200 U/μl)
10 X Ligation Buffer
We recommend that 0.5-1 μl of the ligase is used per 10 μl ligation reaction.
Applications
Cloning of restriction fragments.
Joining linkers and adapters to blunt-ended DNA
Unit Definition
One unit of GB-Clone™ T4 DNA Ligase is defined as the amount of enzyme required to give 50%
ligation of Hind III fragments of λ DNA (5´ DNA termini concentration of 0.12 μM, 300-
μg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1x T4 DNA Ligase
Reaction Buffer.
Storage： Store at -20°C
Storage Buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50% glycerol.
10x T4 Ligase Buffer:
660mM Tris-HCl(PH7.6), 66mM MgCl2, 100mM DTT, 10mM ATP
Physical Purity
The purity is ≥90% as judged by SDS-polyacrylamide gel electrophoresis with
Coomassie® blue staining.
Standard Applications
We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a
fragment into a plasmid vector. These ratios will vary with other types of vectors, for
example, cDNA and genomic cloning vectors. The following example illustrates the
conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA
fragment.
Protocol:
The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1
vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA.
1. Assemble the following reaction in a sterile microcentrifuge tube:
10 X Ligation Buffer 1 μl
vector DNA 100ng
insert DNA 1:1 to 10:1 molar ratio over vector
T4 DNA Ligase 0.5–1μl
Nuclease-Free Water to final volume of 10μl
2. Incubate the reaction at room temperature for 3 hours, or 16°C for 4-18 hours, or
4 °C over night.
Notes:
1. Concatamers may form as ligation products. The extent of concatamer formation
depends on the vector:insert ratio, incubation temperature and incubation time. This
should be taken into account when screening transformants.
