T4 dna ligase, supplied with 10x concentrated ligation buffer that includes atp. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Ligation of bluntended and singlebase pair overhang fragments requires about 50 times as much. Bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. Pdf engineered dna ligases with improved activities in vitro. T4 dna ligase catalyzes the formation of phosphodiester bonds between neighboring 3. B6030 10x t4 dna ligase buffer 10x t4 dna ligase buffer b6030 500mm trishci 100 mm mgcl 2 50 mm dtt 10 mm atp ph 7. D2886 catalyzes the formation of a phosphodiester bond between the terminal 5. To make aliquots of both canvax t4 dna ligase and 5x t4 dna ligase buffer to avoid. Dna fragment and nick joining t4 dna ligase can be used in the insertion of target genes into vectors or in reactions such as the addition of linker to dna. T4 dna ligase is supplied at either 2,000uul or,000uul along with 10x buffer.
At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Structural biochemistryt4 dna ligase wikibooks, open. Dnafragments with blunt or overlapping ends are incubated with t4 dna ligase in 1. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. The primary structure of phage t4 dna ligase has been determined by dna sequencing of a cloned restriction fragment containing its gene, and partial amino acid. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5phosphate and 3hydroxyl ends in double strand dna or rna with blunt or cohesiveend termini. It is a monomeric polypeptide mw 68kda is encoded by. T4 dna ligase for t4 dna ligation, ta cloning, and other. T4 dna ligase is the industry standard for performance and quality at a 1x concentration this reaction buffer assures optimal activity of the enzyme. Atpdependent dna ligases are essential enzymes in both dna replication and dna repair processes. T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm.
It can be used to ligate cohesive or blunt end dna. For details on nebs quality controls for dna ligases, visit our ligase quality page. T7 dna ligase is an atpdependent ds dna ligase from bacteriophage t7. Singlestranded nucleic acids are not substrates for this enzyme. Ligation protocol with t4 dna ligase m0202 protocols. Heat inactivate at 65c for 10 min or at 70c for 5 min. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications.
Mix thoroughly, spin briefly and incubate for 1 hour at 22c. To thaw 5x t4 dna ligase at room temperature and vortex it vigorously to mix the components. T4 dna ligase is the industry standard for performance and quality. T4 dna ligase inactivation t4 dna ligase can be completely inactivated by a 10 min incubation at 65c. Ligated dna is suitable for direct use in transformation experiments. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Dna ligase is a specific type of enzyme, a ligase, ec 6.
Primary structure and genetic organization of phage t4 dna. Here we report that this ligase, unlike escherichia coli dna ligase, taq dna ligase. T3 dna ligase is also active in buffers without peg 6000, such as our t4 dna ligase buffer and nebuffers 14, for applications in which peg 6000 is detrimental. Here we report a functional characterization of the t4 dna ligase. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Templateindependent ligation of singlestranded dna by t4. Dna ligase i is more effective at bluntend joining than mam malian dna ligases i1 and 111 but is less efficient in this regard than bac teriophage t4 dna ligase. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna using atp as a cofactor. T4 dna ligase recombinant form of the enzyme from t4 phage. 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 extent of ligation can be determined by estimation of the relative intensity of fluorescence in the gel bands of substrate and product. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. T4 dna ligase competitor study nuclease contamination t4 dna ligase from multiple suppliers was tested. Joining linkers and adapters to bluntended 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. Several wild type dna ligases with known endjoining activity hlig3, t3 dna ligase, pbcv1 dna ligase, t7 dna ligase, and e. Sample material dephosphorylated dna with either blunt or sticky ends.
Ratio of vector arms to dna the ratio of vector arms to insert dna should be approx. The t4dnl t4 dna ligase and two rna ligases t4rnl1 t4 rna ligase 1 and t4rnl2 join a diverse array of substrates, including nicks that are present in doublestranded nucleic acids. Dna ligase reconnects dna strands when they are broken. Depending on the dna concentration in the reaction, the ligation products will be either circular if the dna concentration is low or concatemeric if the dna concentration is high.