The dna ligation kit contains the reagents necessary to increase the consistency of ligations. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Dna ligase is a minimum of 95% as determined by sds page analysis using coomassie blue detection. T4 dna ligase catalyzes the joining of two strands of dna between the 5. The enzyme will not join singlestranded nucleic acids. Linear vector dna 20100 ng insert dna over vector thermo scientific 10x t4 dna ligase buffer 2. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in,biological,biology supply,biology supplies,biology product. To dilute t4 dna ligase that will subsequently be stored at. Learn more about how this product is being used in the product citation tool.
For bacterial transformation we offer a choice of competent cells, including our unique single step krx strain. A similar structure, that of t7 dna ligase, has been solved subramanya et al. Dna ligase 1 utilizes adenosine triphosphate atp to catalyze the energetically favorable ligation events in both dna replication and 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. T4 dna ligase ligase dna ligation promega corporation. Eukaryotic dna ligase 1 catalyzes a reaction that is chemically universal to all ligases. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Promega enzyme resource guide, cloning enzymes, br075b. Ligafast rapid dna ligation system protocolpdf promega. The ligafast system is designed to eliminate any further. Ligations using promega t4 dna ligase see product info sheet for more information. The dna ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. Dna ligations are performed by incubating dna fragments with appropriately linearized cloning vectors in the presence of buffer, atp, and dna ligase. Wizard genomic dna purification kit protocol promega.
After ligation, the insert dna is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation. Product has been classified in accordance with the hazard criteria of the cpr and the msds contains all of the information required by the cpr. Promega enzyme resource guide 8 two ligases introduction dna ligases are primarily responsible for joining the gaps that form in dna during replication i. Promega for quality control of lambda vectors with sticky ends. T4 dna ligase reaction buffer t4 dna ligase is the industry standard for performance and quality. What is supplied with the t4 dna ligase reaction buffer pack. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3.
Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. Since dna ligase ii appears to be derived from dna ligase iii by a proteolytic mechanism, the three lig genes can account for the four biochemically distinct dna ligase activities, dna ligases i, ii, iii and iv, that have been purified from mammalian cell extracts. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in a duplex molecule but. We enable science by offering product choice, services, process excellence and our people make it happen. Ligafasttm rapid dna ligation system product information. Dna ligase iv is a component of the nhej complex and participates in the repair of dna doublestrand breaks that arise during dna damage, such as from ionizing radiation, or the endogenous recombination process. In the manual for the t4 dna ligase i use promega, it is indicated that for longer incubation times a low temperature is preferred. For conventional cloning of dna fragments, we provide a variety of restriction enzymes and t4 dna ligase. Do not exceed the recommended amount of t4 dna ligase in the rection mixture. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dnarna hybrids 1. Cloning and dna markers tools that modify nucleic acids provide the foundation for many molecular biology techniques such as subcloning. 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. To dilute t4 dna ligase that will subsequently be stored at 20c, 50% glycerol storage buffer.
A protocol for isolation of genomic dna from white blood cells, tissue culture cells, animal and plant tissue, yeast, grampositive and gramnegative bacteria. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Dna ligases are primarily responsible for joining the gaps that form in dna during replication. No conversion of covalently closed circular dna to product. T4 dna ligase rapid the enzyme efficiently joins blunt and.
The recommended concentration of peg 4000 in the ligation reaction mixture is 5% wv. T4 dna ligase bluewhite cloning qualified protocol promega. T4 dna ligase is used in molecular cloning experiments to ligate join the ends of cloned dna fragments, ligase is commonly to insert dna into a cloning vector. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 dna ligase is active in pcr and restriction polyethylene glycol peg greatly increases the ligation efficiency of bluntend dna ligation. Instructions for use of products m1801, m1804, m1794. Each promega product is shipped with documentation.
Promega products are warranted to meet or exceed the. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. T4 dna ligase can be used to join dna fragments with staggered or blunt ends and to repair nicks in doublestranded dna having 3hydroxyl and 5phosphate ends. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a. T4 dna ligase is provided with 10x reaction buffer. Examples include protein expression, mutagenesis, gene analysis and structurefunction relationships. Toll free in the usa 8003569526 telephone 6082744330 internet.
L this protocol is for the dna insert ligation stickyend and bluntend into vector dna. A confidential page 4 of 5 section xi toxicological information conditions aggravated target organs. Dna ligase is a specific type of enzyme, a ligase, ec 6. Ligases are enzymes that seal the gap in the phosphodiester backbone of the dna. For other cloning applications we offer t4 dna kinase, calf alkaline phosphatase and other modifying enzymes. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Learn more about t4 dna ligase 10x buffer, promega. 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. T4 dna ligase is used in molecular cloning experiments to ligate join the. This reaction, called ligation, is performed by the t4 dna ligase enzyme. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dnarna hybrids. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5.
Manual, second edition, cold spring harbor laboratory press, cold spring harbor. The wizard genomic dna purification kit is based on a fourstep process. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. The wizard genomic dna purification kit is designed for isolation of dna from white blood cells, tissue culture cells and animal tissue,plant tissue, yeast, grampositive and gramnegative bacteria. 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. These ligation conditions have been developed using promega bluewhite cloningqualified t4 dna ligase. Also catalyzes the joining of rna to either a dna or rna strand. These features are consistent with dna ligase iv knockout mice, which are characterized by defective lymphogenesis and neurogenesis. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1.
T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. T4 dna ligase buffer pack from promega description 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. These ratios will vary with other types of vectors, for example, cdna and genomic cloning vectors. The roche t4 dna ligase manual im using says ligation should be kept for 16h. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful.
Ligase from t4infected escherichia coli and promega t4 dna ligase buffer. L t4 dna ligase 5 u water, nucleasefree total volume 20. They help in completing the gap between okazaki fragments during dna replication, and also the gaps formed during various dna repair mechanisms and also in recombinan. The enzyme has also been shown to catalyze the joining of rna. The first step in the purification procedure lyses the cells and the. T4 dna ligase rapid the enzyme efficiently joins blunt. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. Signs and symptoms of excessive exposure may be central nervous system effects and increased blood sugar levels.
These ratios will vary with other types of vectors, for example, cdna and genomic. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. A protocol for combining two strands of dna between the 5. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5.
What is the composition of the t4 dna ligase reaction buffer. Lig1 encodes dna ligase i, which functions in dna replication and the base excision repair process. As with t4 dna ligase, blunt end ligation is enhanced by the addition of peg 6000 to the reaction. Purified dna is suitable for amplifications, restriction enzyme digestion and membrane hybridizations e. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. It will catalyze the formation of a phosphodiester bond between adjacent 5. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. The addition of polyethylene glycol peg to ligation reactions can promote ligation of bluntended fragments by macromolecular crowding 2.