T4 dna ligase is mostly used in the joining of dna molecules with compatible cohesive termini, or bluntended, doublestranded dna to one another, or to synthetic linkers. The ligase 10x buffer supplied with this enzyme has a composition of 300mm. One weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1. The human lig3 gene encodes atpdependent dna ligases that seal interruptions in the phosphodiester backbone of duplex dna.
The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Dna ligase iv is part of the nonhomologous end joining. The formation of a phosphodiester bond between juxtaposed 5. No conversion of covalently closed circular dna to product. The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. Dna ligase is a specific type of enzyme, a ligase, ec 6. Saltt4 dna ligase will join cohesive end termini at salt concentrations as high as 300 mm without any loss in activity. Sitedirected mutagenesis using pfu dna polymerase and t4. T4 dna ligase is active in pcr and restriction polyethylene glycol peg greatly increases the ligation efficiency of bluntend dna ligation.
T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Learn more about how this product is being used in the. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in a duplex molecule, but will not join singlestranded. 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 sequence analysis of the protein. Hyonemyong eun, in enzymology primer for recombinant dna technology, 1996. T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm. The enzyme was stable for 1 week at 37 c, its activity dropped by 50% within 2 days at 65 c. Ligation produced only linear dna because the dna concentration was too high. Saltt4 dna ligase is insensitive to salt carried over from other reaction components. One unit is defined as the amount of enzyme required to give 50% ligation of hind iii fragments of dna 5. Full text get a printable copy pdf file of the complete article 1. A similar structure, that of t7 dna ligase, has been solved subramanya et al. Readytogo t4 dna ligase from ge healthcare, formerly amersham biosciences 9. The recommended concentration of peg 4000 in the ligation.
Dna ligases are enzymes that can form a phosphodiester bond at a singlestrand break in dna, a reaction between a 3. Therefore, we have aligned our dna ligase production methodologies with an expansive series of quality controls below, and are constantly testing our dna ligases in house with our internal research. 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 from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3overhang or 5 overhang containing oligonucleotides.
T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. T4 dna ligase catalyzes the joining of two strands of dna between the 5. From the kinetics of the reaction and from the absence of nicked dna in the final products it was inferred that dna ligase acts processively through a nickingclosing mechanism. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. T4 dna ligase is used in molecular cloning experiments to ligate join the. The position of the conserved boxes see text is indicated. The performance of this buffer depends on the integrity of the atp.
The reaction that involves blundended dna is slower than the other reactions, but the rate of ligation can be accelerated by adding 150 to 200 mm of nacl along with a low. One microliter approximately 100 ng of each pcr product was directly used for bluntend ligation 1 h at room temperature, 1. Singlestranded nucleic acids are not substrates for this enzyme. The most commonly used is the t4 dna ligase method. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of.
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. Identification of the substancepreparation and of the companyundertaking product name t4 dna ligase product no m0202 recommended use of the chemical and restrictions on use. T4 dna ligase 10x t4 dna ligase buffer 50% peg solution notes binding of t4 dna ligase to dna may result in a band shift in agarose gels. Ligation of bluntended and singlebase pair overhang fragments requires about 50 times as much enzyme to achieve. Invitrogen t4 dna ligase 1u l ideal for cloning bluntend or cohesiveend ligation and adding linkers or adapters to bluntended dna manufacturer. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. Dna ligase iv deficiency is a rare primary immunodeficiency, lig4 syndrome, often associated with other systemic features. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. Similarly to mammalian dna ligase i, t4 dna ligase can relax supercoiled dna in an ampdependent reaction that could be referred to as the reverse reaction 7,8. Schematic representation of the different t4 dna ligase mutants expressed in li as histagged proteins a.
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. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and returned immediately to 20 c after use. T4 dna ligase ligase dna ligation promega corporation. The strands to be ligated need to be hybridized and accurately paired, with no gap, to a complementary dna strand. What are some potential problems with the ligation. Feb 25, 1991 amounts of t4 dna ligase in large molar excess to dna template and ligated product are necessary to achieve high yields. T4 dna ligase rapid the enzyme efficiently joins blunt. Safety data sheet document type aghs osha ghs revision date 21jul2016 version 3 1. An optimized blend of a thermostable dna ligase and a proprietary additive, hifi taq dna ligase efficiently seals nicks in dna with unmatched high fidelity. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Dna ligase 3 is an enzyme that, in humans, is encoded by the lig3 gene. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. It will catalyze the formation of a phosphodiester bond between adjacent 5. Catalyzes the ligation of blunt or cohesiveend dna fragments in only 5 minutes at room temperature 25c, and pcr fragments with a overhangs in 15.
Learn more about how this product is being used in the product citation tool. Dna ligase iv is the nhejspecific ligase grawunder et al. T4 dna ligase rapid the enzyme efficiently joins blunt and. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5 phosphate and a 3 hydroxyl groups of duplex dna or rna. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. The following protocol is for rapid ligation of cohesive ends. Structural biochemistryt4 dna ligase wikibooks, open books. This enzyme will ligate dna from bluntend and cohesiveend termini including sticky ends for ta cloning as well as repair single stranded nicks in duplex dna, rna, or dna rna hybrids. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. 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. Ligase master mix instant stickyend master mix electroligase t4 dna ligase hit4 dna ligase saltt4 dna ligase quick ligation kit t3 dna ligase t7 dna ligase hifi taq dna ligase taq dna ligase 9n dna ligase nebnext quick ligation module splintr ligase li dna ligase. T4 dna ligase for t4 dna ligation, ta cloning, and other.
T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Set up the following reaction in a microcentrifuge tube on ice. Taq dna ligase is a thermostable ligase that catalyzes the formation of a phosphodiester bond between the 5. The ligase 10x buffer supplied with this enzyme has a composition of. The enzyme efficiently joins blunt and cohesive ends and. Ligation protocol with t4 dna ligase m0202 protocols. 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. 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. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase 1.
Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. The third utilizes the enzyme terminal deoxynucleotidyl transferase to synthesize homopolymeric 3. In addition to t4 dna ligase and the insert and vector dnas, the following reagents and equipment are required for the protocols described below. The journal of biological chemistry 0 1984 by the american society of biological chemists, inc. Dna helicase, rna primase, dna polymerase iii, exonucleases, dna polymerase i, dna ligase function of dna sores cells genetic info that is used to make proteins. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. It can be used to ligate cohesive or blunt end dna fragments. What are some potential problems with the ligation reaction. A novel dna joining activity catalyzed by t4 dna ligase. Singlestranded nucleic acids are not substrates for thi.
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