T4 DNA Ligase 200uL
Overview
T4 DNA Ligase catalyzes the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA using ATP as a coenzyme. Both blunt and cohesive end DNA ligation, as well as single-stranded nick repair of DNA, RNA and DNA/RNA, are possible via the T4 DNA ligase. 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.
Biological Source
- E. coli strain expressing a recombinant clone.
Molecular Weight
- 68kDa.
Requirements
Mg , ATP and DTT. The optimum concentration of Mg is 10mM. Mn may be substituted for Mg but is only 25% as effective as Mg .
Inhibition
50% inhibition by greater than 150mM NaCl (activity measured at nicks). Other inhibitors include 0.2M K , Cs , Li , NH and 1mM spermine.
Inactivation
- Heat to 70°C for 10 minutes.
Contents
- T4 DNA Ligase (200 U/μl).
- 10 X Ligation Buffer.
Applications
- Cloning of restriction fragments.
- Joining linkers and adapters to blunt-ended DNA.
Unit Definition
One unit of 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 MgCl , 100mM DTT, 1mM 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.
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