T4 DNA Ligase

Name: T4 DNA Ligase
SKU: 1380
Availability: InStock

Price range: $201.00 through $335.00

Categories: Cloning Kits & Cloning Related Enzymes, Enzymes, T4 Enzymes & Related Products.

Description

Intact Genomics T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA. This enzyme joins DNA fragments with either cohesive or blunt termini and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).

Applications

• Cloning of restriction enzyme generated DNA fragments
• Cloning of PCR products
• Next-gen library preparation
• Joining linkers and adapters to cohesive or blunt-ended DNA
• Nick repair in duplex DNA, RNA or DNA/RNA hybrids
• Self-circularization of linear DNA

Purity

The physical purity of Intact Genomics T4 DNA Ligase is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).

Comparison Testing

Intact Genomics T4 DNA displays significantly higher ligation efficiency than the nearest competitor.

Product Source
E. coli strain expressing a recombinant clone

Product Includes

T4 DNA Ligase
10x T4 DNA Ligase Reaction Buffer

Storage Buffer

50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 ºC

1x T4 DNA Reaction Buffer
50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 7.5 @ 25 ºC

Unit Definition
One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (250 ng/µl) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA ligase reaction buffer.

Inhibition and Inactivation

Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50 mM.
Inactivated by heating at 70 °C for 15 min or by addition of EDTA.

Quality Control Assays

Endonuclease Activity (Nicking)

1 µg of supercoiled plasmid DNA is incubated with 20 units of T4 DNA Ligase in 1x Ligase buffer for 2 hours at 37 ºC. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel. No visible nicking or cutting of DNA was found.

Functional Assay

DNA Ligase functional efficiency is tested in cloning assays.3212 3216 3217

Additional information

Units	20,000 units (400 units/µl), 100,000 units (400 units/µl), 100,000 units (2000 units/µl), 400,000 units (2000 units/µl)

Set up reaction buffer in a microcentrifuge tube on ice. Use a molar ratio of 1:3 vector to insert DNA.
Gently mix the reaction and centrifuge briefly.
For cohesive ends, incubate at room temperature for 10 min or 16 ºC for overnight.
For blunt ends, incubate at room temperature for 2 hours or 16 ºC for overnight.
Heat inactivate at 70 ºC for 15 min.
Cool on ice and transform 2 µl of the reaction into 50 µl competent cells.