T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between doublestranded DNAs with 3 hydroxyl and 5 phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes. How can the answer be improved? Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'phosphate and 3'hydroxyl termini in duplex DNA or RNA.
The enzyme repairs singlestrand nicks in duplex DNA, RNA, or DNARNA hybrids. CERTIFICATE OF ANALYSIS T4 DNA Ligase# EL0015 200u Lot: Quality guaranteed: transformation of bacterial cells with DNA. Activity in Fermentas REase Buffers, Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
vector DNA 100ng insert DNA 17ng Ligase 10X Buffer 1l T4 DNA Ligase (Weiss units) 0. 11u NucleaseFree Water to final volume of 10l 2. Incubate the reaction at: room temperature for 3 hours, or 4C overnight, or 15C for 418 hours. Notes: 1. There is considerable latitude in the temperature and time needed for successful ligations.
Rapid Ligation of Cohesive Ends (5min ) for Plasmid Cloning of DNA Fragments: Note: The following protocol is for rapid ligation of cohesive ends. For rapid ligation of blunt ends, use T4 DNA Ligase, Cat no.which has a concentration of 5 Ul.
This higher concentration is required for rapid ligation of blunt ends. T4 DNA Ligase catalyzes the formation of phosphodiester bonds between neighboring 3hydroxyl and 5phosphate ends in doublestranded DNA. Singlestranded nicks in doublestranded DNA are also closed by T4 DNA Ligase. T4 DNA Ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3overhang or 5 overhang containing oligonucleotides.
The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. T4 DNA Ligase catalyzes the joining of two cohesive or bluntended strands of DNA between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides.
The enzyme will not join singlestranded nucleic acids. If more than 2 Weiss U of T4 DNA ligase is used in 20 L reaction mixture, it is necessary to purify DNA (by spin column or chloroform extraction) before conventional T4 DNA ligase reactions (2. 8 Weiss units of T4 DNA Ligase, in standard ligation buffer, incubated at 16C for 16 hours). 25 ng of EcoR Idigested pBR322 plasmid DNA (4361 bp) and 250 ng of EcoR Idigested, dephos Intact Genomics T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl 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 DNARNA hybrids (1).