Can T4 DNA ligase be inactivated?
Standard T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes.
What is the function of T4 DNA ligase?
T4 DNA Ligase catalyzes the joining of two cohesive- or blunt-ended strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides. The enzyme will not join single-stranded nucleic acids.
What is T4 DNA ligase how is it used to clone DNA?
T4 DNA Ligase is a ligation enzyme that can be used to join DNA fragments by catalyzing the formation of phosphodiester bonds between juxtaposed 5′ phosphate and 3′ hydroxyl termini in double-stranded DNA using ATP as a coenzyme.
What is the definition of one unit of T4 DNA ligase?
Unit Definition: (Cohesive End Ligation Unit): One NEB unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM [300 µg/ml]) in 20 µl of 1X T4 DNA Ligase Reaction Buffer in 30 minutes at 16°C.
How is T4 DNA ligase used in DNA ligation?
T4 DNA Ligase catalyzes the joining of two cohesive- or blunt-ended strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides. The enzyme will not join single-stranded nucleic acids. T4 DNA Ligase is provided with 10X Reaction Buffer: 300mM Tris-HCl (pH 7.8 at 25°C), 100mM MgCl 2, 100mM DTT and 10mM ATP.
How long can you Heat inactivate a T4 ligase?
Standard T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes. Do not heat inactivate the Quick Ligation Kit or ligase master mixes. Add between 1-5 µl of ligation mixture to competent cells for transformation. Extended ligation with PEG causes a drop off in transformation efficiency (Quick Ligation Kit).
Can You ligate T4 DNA at room temperature?
Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP. Room Temperature Ligation. For convenience, ligations may be done at room temperature (20-25°C).
How does ligafast ( TM ) rapid DNA ligation system work?
Rapid ligation is based on the combination of T4 DNA Ligase with a unique 2X Rapid Ligation Buffer. The LigaFast™ System is designed to eliminate any further purification prior to transformation of ligated DNA. The specially formulated 2X Rapid Ligation Buffer requires no ATP or Mg2+addition prior to use.
