Call Us Today! 1-855-835-7172|sales@intactgenomics.com

T4 DNA Ligase

$60.00$299.00


    200 Weiss Units (low conc.) - $60.00 (Cat.# 3210 )

    500 Weiss Units (low conc.) - $95 (Cat.# 3212 )

    500 Weiss Units (high conc.) - $95.00 (Cat.# 3216 )

    2,000 Weiss Units (high conc.) - $299.00 (Cat.# 3217 )

Categories: , .

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 enzymes joins DNA fragments with either cohesive or blunt termini as well as repair 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).

1a

Comparison Testing

Intact Genomics T4 DNA Ligase displays up to 3-5X higher ligation efficiency than the nearest competitor.

T4 DNA Ligase

Additional information

Units

200 Weiss Units (low conc.), 500 Weiss Units (low conc.), 500 Weiss Units (high conc.), 2,000 Weiss Units (high conc.)

Product Source
E. coli strain expressing a recombinant clone

Product Includes

  1. T4 DNA Ligase
  2. 10x T4 DNA Ligase Reaction Buffer (w/o ATP)
  3. 10 mM ATP

Storage Buffer

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

10x T4 DNA Ligase Reaction Buffer (w/o ATP)

500 mM Tris-HCl, 100 mM MgCl2, 100  mM DTT, pH 7.5 @ 25 ºC

Note
10x T4 DNA ligase buffer does not contain ATP. You need to add ATP separately.

Unit Definition

One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P from pyrophosphate into Norit-absorbance material in 20 minutes under standard assay conditions.

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.

  1. Set up reaction buffer in a microcentrifuge tube on ice. Use a molar ratio of 1:3 vector to insert DNA.
  2. 3aGently mix the reaction and centrifuge briefly.
  3. For cohesive ends, incubate 16 ºC for overnight or at room temperature for 30 min.
  4. For blunt ends, incubate 16 ºC for overnight or at room temperature for 2 hrs.
  5. Heat inactivate at 70 ºC for 15 min.
  6. Cool on ice and transform 2 µl of the reaction into 50 µl competent cells.

 

Reference

  1. Engler, M.J and Richadson, C.C (1982) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, NY.

 

T4 DNA Ligase

You may also like…