T4 dna ligase rapid the enzyme efficiently joins blunt and. Genetic engineering is a modern biotechnology technique which is used to modify the genetic makeup of an organism by adding new traits in to it and there by produce new variety of organ. The infection with a t4 mutant, t4awh39x which is defective in the dna ligase gene, enables preparation of rna ligase free of dna ligase 1. Shop online for a wide selection of invitrogen t4 dna ligase 1 ul ideal for cloning bluntend or cohesiveend ligation.
The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3. Ligation by t4 dna ligase was found to be dependent on the. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. 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. 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. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. We used t4 dna ligase and the four best fusion proteins to ligate an arbitrary, bluntended dsdna fragment into a standard cloning vector puc18 that had been linearized by digestion with smai. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. It can be used to ligate cohesive or blunt end dna fragments. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna, rna or dna rna hybrids.
Intact genomics t4 dna ligase displays significantly higher ligation efficiency than the nearest competitor. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Engineered dna ligases with improved activities in vitro. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i. Please remember to supplement the reaction with 1 mm atp final concentration.
T4 dna ligase is an enzyme that is encoded by the bacteriophage known as t4. Singlestranded nucleic acids are not substrates for thi. 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. The molecule has a mr of 55,230, and contains 487 amino acids. Heat inactivate at 65c for 10 min or at 70c for 5 min. 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. For example, dna fragments are cut with restriction enzymes and then recombined with dna ligase. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. E invitrogen, carlsbad, ca, usa was used for all cloning and protein expression. For details on nebs quality controls for dna ligases, visit our ligase quality page. The free ends of the input dna molecules need not be homologous in order for efficient recombination. 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. Bluntta ligase master mix, t4 dna ligase, 10x t4 dna ligase buffer.
Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Dna ligase is a specific type of enzyme, a ligase, ec 6. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna, rna or dnarna hybrids. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5. Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. The temperature dependence of the t4 dna ligasecatalyzed joining of plasmid dna linearized by the action of haeiii, ecori and psti restriction endonucleases has been investigated by electron. Different substrate specificities of the two dna ligases of mammalian. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. To test the onestep subcloning strategy 50 ng of each uncut plasmid was added to a single tube together with 2.
Invitrogen t4 dna ligase 1u l ideal for cloning bluntend or cohesiveend ligation and adding linkers or adapters to bluntended dna. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration. L s33102 egel ex starter kit, 1% 1 kit g6511st technical support. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. Atpdependent dna ligases are essential enzymes in both dna replication and dna repair processes. Nov 05, 2008 to test the onestep subcloning strategy 50 ng of each uncut plasmid was added to a single tube together with 2. T4 dna ligase for t4 dna ligation, ta cloning, and other. 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 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 nucleic. Wholegenome amplification by adaptorligation pcr of. Use of t4 phage mutants dorega enables one to overproduce the ligase to as much as sevenfold over the amount of wild type or 0. Dnahind iii fragments analyzed by agarose gel electrophoresis. Singlestranded nucleic acids are not substrates for this enzyme.
Dna ligases are a useful tool in generating recombinant dna sequences. Pdf templateindependent ligation of singlestranded dna by t4. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. Dna ligase synonyms, dna ligase pronunciation, dna ligase translation, english dictionary definition of dna ligase. Primary structure and genetic organization of phage t4 dna. Bamhi site of the ptrchisa expression vector invitrogen. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Pdf functional characterization of the t4 dna ligase. Pdf atpdependent dna ligases are essential enzymes in both dna replication and dna repair. 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. Note t4 dna ligase is active in pcr and restriction. One nterminal and two cterminal deletion mutants were expressed in escherichia coli as histidinetagged proteins. Here we report a functional characterization of the t4 dna ligase. A one pot, one step, precision cloning method with high.
Efficient assembly of very short oligonucleotides using t4 dna ligase. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. 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. Pdf t4 dna ligase is one of the workhorses of molecular biology and used in. Here and below, m denotes a 25bp dna ladder invitrogen. M270 streptavidin dynabeads invitrogen were washed three times with. The physical purity of intact genomics t4 dna ligase is. Ambion nucleasefree water was obtained from life technologies.
Structural biochemistryt4 dna ligase wikibooks, open books. Mix thoroughly, spin briefly and incubate for 1 hour at 22c. Great for most common ligation applications including ta cloning. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1.
Interaction of bacteriophage t4 rna and dna ligases in joining of. Ligation protocol with t3 dna ligase m0317 ligation protocol with t7 dna ligase m0318 ligation protocol using splintr ligase m0375 e. The temperature dependence of the t4 dna ligase catalyzed joining of plasmid dna linearized by the action of haeiii, ecori and psti restriction endonucleases has been investigated by electron. In most laboratories, the primary use for t4 dna ligase remains in the construction of recombinant plasmids.
Blockit u6 rnai entry vector kit thermo fisher scientific. Pdf dna cloning without restriction enzyme and ligase. A similar structure, that of t7 dna ligase, has been solved subramanya et al. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5. Ligafasttm rapid dna ligation system certificate of. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. A nucleic acid that carries the genetic information in cells and some viruses, consisting of two long. T4 dna ligase 250 units 15224041 t4 polynucleotide kinase 1,000 u 18004028 ultrapure low melting point agarose 100 g 16520100 trackit 1 kb plus dna ladder 100 applications 10488085 sybr safe dna gel stain 10,000x 400.
A highthroughput assay for the comprehensive profiling of dna. Dna ligase definition of dna ligase by the free dictionary. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of. Engineered dna ligases with improved activities in vitro protein. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dnarna hybrids 1. An additional mutant bore a substitution of lys159 in the active. The protein encoded by this gene is a dna ligase that joins singlestrand breaks in a doublestranded polydeoxynucleotide in. Dna ligase reconnects dna strands when they are broken. The free end is designed with a variable 5 overhang, complementary to labeled oligonucleotides used in ligation. 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.