GB-Clone™ FAST T4 DNA Ligase is designed for the efficient ligation of cohesive-ended DNA inserts
into plasmid vectors in just 5-10 minutes (blunt-ended inserts in as little as 15-30 minutes).
FAST T4 DNA Ligase catalyzes the joining of two strands of DNA between the
5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides in either a
cohesive-ended or blunt-ended configuration. The enzyme has also been shown to
catalyze the joining of RNA to either a DNA or RNA strand in a duplex molecule but will not
join single-stranded nucleic acids.
Biological Source: E. coli strain expressing a recombinant clone.
Molecular Weight: 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: FAST T4 DNA Ligase (400 U/μl)
2 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 FAST 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℃
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.
2X T4 DNA Ligase Buffer: 60mM Tris-HCl (pH 7.8), 20mM MgCl2, 20mM DTT, 2mM ATP and 10% PEG.
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:
vector DNA 100ng
insert DNA 17ng
2 X Ligation Buffer 5μl
T4 DNA Ligase 0.5–1μl
Nuclease-Free Water to final volume of 10μl
2. Incubate the reaction at room temperature for 5-10 minutes for cohesive-ended
ligations, or 15-30 minutes for blunt-ended ligations.
Notes:
1. Ligation reactions performed using the 2 X Ligation Buffer do not need to be cleaned up
before transformation.
2. 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.