Great for most common ligation applications including ta cloning. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Pdf functional characterization of the t4 dna ligase. 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. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. For details on nebs quality controls for dna ligases, visit our ligase quality page. Please remember to supplement the reaction with 1 mm atp final concentration. T4 dna ligase is the industry standard for performance and quality. There is considerable latitude in the temperature and time needed for successful ligations. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. T4 dna ligase structure reveals a prototypical atpdependent.
Invitrogen t4 dna ligase 1u l ideal for cloning bluntend or cohesiveend ligation and adding linkers or adapters to bluntended dna. Reactions were incubated for 10 min at 16c, before being stopped. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. It is not appreciably active on bluntended substrates.
Dna ligase reconnects dna strands when they are broken. For blunt ends, use 1 l of t4 dna ligase in a 20 l reaction for 2 hours or 1 l high concentration t4 dna ligase for 10 minutes. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. If the dna ligase is incubated with cxpiatp, the enzyme. T4 dna ligase catalyzes the ligation of two rna substrates that are precisely aligned in a fully basepaired rna dna heteroduplex, whereas t4 rna ligase is used to join two singlestranded rnas in. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and.
Sitedirected mutagenesis using pfu dna polymerase and t4. T4 dna ligase is used for the joining of dna molecules with compatible cohesive sticky termini, joining of blunt ended double stranded. Ligafasttm rapid dna ligation system certificate of. It plays a role in repairing singlestrand breaks in duplex dna in living organisms, but some forms such as dna ligase iv may specifically repair doublestrand breaks i. The most commonly used is the t4 dna ligase method. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Efficient ligation of dna on rna templates using a mutated t4. Vaccinia virus encodes a 552 amino acid dna ligase that has a nicksensing function.
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. It can be used to ligate cohesive or blunt end dna fragments. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna strands with 3 hydroxyl and 5 phosphate termini in the presence of atp. Each microgram of cut plasmid is ligated with 4 units of t4 dna ligase. New england biolabs t4 dna ligase reaction buffer 50 mm trishcl, 10 mm mgcl 2, 10 mm dithiothreitol, 1 mm atp, ph 7. Set up the following reaction in a microcentrifuge tube on ice. Sitedirected mutagenesis using pfu dna polymerase and t4 dna. T4 dna ligase for t4 dna ligation, ta cloning, and other. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Quicklink dna ligation kit lig2 technical bulletin sigmaaldrich. Investigation of the ligation efficiency of ndei digested.
The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Bacteriophage t4 dna ligase is the first atpdependent ligase enzyme to be. T4 dna ligase competitor study nuclease contamination 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. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. Ligation protocol with t4 dna ligase m0202 protocols. In order to study its structure and activity in vitro we have undertaken the recombinant production and puri. Ligation of dna fragments with blunt ends vectorinsert ligation.
Gently mix the reaction by pipetting up and down and microfuge briefly. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. Singlestranded nucleic acids are not substrates for this enzyme.
Pdf atpdependent dna ligases are essential enzymes in both dna replication and dna repair. On the basis of an analogous approach to dna 59adenylation using t4 dna ligase chiuman and li 2002 and our own procedure for rna 59adenylation with t4 rna ligase silverman 2004, here we. T4 dna ligase the enzyme efficiently joins blunt and. T4 dna ligase catalyzes the ligation of two rna substrates that are precisely aligned in a fully basepaired rnadna heteroduplex, whereas t4 rna ligase is used to join two singlestranded rnas in. Generation of intermediate plasmids for the investigation of. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. The prototype of these dna ligases is the most commonly used t4 dna ligase. For the t4 dna ligase variants, each 10 l activity assay contained 150 ng of this dna substrate. One unit of enzyme and one mircolitre of ligase buf fer were used in each r eaction. Dna ligase is a specific type of enzyme, a ligase, ec 6. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. The genome of the pathogenic psychrophile aliivibrio salmonicida encodes one such putative atpdependent dna ligase 10.
It is also interesting that t4 ligase is able to catalyze the joining of bluntended. Atpdependent dna ligases are essential enzymes in both dna replication and. T4 dna ligase the enzyme efficiently joins blunt and cohesive. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature.
Recombinant expression and purification of an atpdependent. T4 dna ligase rapid the enzyme efficiently joins blunt and. Invitrogen 15224017 view more versions of this product. Engineered dna ligases with improved activities in vitro. Invitrogen t4 dna ligase 1u l ideal for cloning bluntend or cohesiveend ligation and adding linkers or adapters to bluntended dna manufacturer. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master mix. Bamhi site of the ptrchisa expression vector invitrogen.
Pdf templateindependent ligation of singlestranded dna by t4. Learn more about how this product is being used in the product citation tool. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. Generation of intermediate plasmids for the investigation. Singlestranded nucleic acids are not substrates for thi. The enzyme efficiently joins blunt and cohesive ends and repairs singlestranded nicks in duplex dna, rna or dnarna hybrids. One microliter approximately 100 ng of each pcr product was directly used for bluntend ligation 1 h at room temperature, 1. The crystal structure of this 41kda enzyme has been resolved and shows that the enzyme comprises of two domains with a deep cleft between them 3. The ligafast rapid dna ligation system is designed for the efficient ligation of cohesiveended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. Pdf t4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological. D2886 catalyzes the formation of a phosphodiester bond between the terminal 5.
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