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. Ligation protocol with t4 dna ligase m0202 protocols. 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. Ligase from t4infected escherichia coli and promega t4 dna ligase buffer. Singlestranded nucleic acids are not substrates for. Promega ligafast rapid dna ligation system 2x rapid. Mix thoroughly, spin briefly and incubate for 1 hour at 22c. Dna ligase is a specific type of enzyme, a ligase, ec 6. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. Please remember to supplement the reaction with 1 mm atp final concentration. The optimal insert to vector dna ratio, is usually between 2. Dna cloning without restriction enzyme and ligase article pdf available in biotechniques 276.
Ligation efficiency is also contingent on the integrity of the cohesive ends of the fragments being ligated. Oct 26, 2015 rna and dna adenylation by t4 rna ligase 1. L of purified pcr product from the pcr cleanup plate, 2. Efficient dna ligation in dnarna hybrid helices by. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. Singlestranded dna ligation with t4 dna ligase and circligase. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. 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 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. Bacteriophage t4 dna ligase is the ligase most commonly used in the. The roche t4 dna ligase manual im using says ligation should be kept for 16h. Higher concentrations of dna reaction components will result in a higher rate of reaction. Note that the table shows a ligation using a molar ratio of 1.
Singlestranded dna library preparation from highly degraded. The most commonly used is the t4 dna ligase method. For this we utilized t4 dna ligase promega under the standard manufacturers conditions. 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. T4 dna ligase catalyzes the joining of two strands of dna between the 5. 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. A previously reported method used double stranded t4 dna ligase to perform enzymatic adenylation 9. 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 can be used to join dna fragments with staggered or blunt ends. L of ligation master mix to each well of a new pcr plate. Gently mix the reaction by pipetting up and down and microfuge briefly. To test for endonuclease activity, 1g of type i supercoiled plasmid dna is incubated with 20 units of t4 dna ligase in 1x rapid ligation buffer for 16 hours at 37c. Singlestranded nucleic acids are not substrates for this enzyme. T4 dna ligase ligase dna ligation promega corporation.
Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. A pool of 60 nt acceptor oligonucleotides 60n were ligated to 10 pmol of a 3. Splint ligation of rna, whereby specific rna molecules are ligated together, can be carried out using t4 dna ligase and a bridging dna oligonucleotide complementary to the rnas. There is considerable latitude in the temperature and time needed for successful ligations. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. 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.
Heat inactivate at 65c for 10 min or at 70c for 5 min. It is based on two rarecutting restriction enzymes, sgfi and pmei, and provides a rapid, efficient and highfidelity way to transfer proteincoding regions without the need to resequence. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. 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. Ligation products were visualized on a 10% denaturing. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Promega ligafast rapid dna ligation system 2x rapid ligation. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 dna ligase is provided with 10x reaction buffer. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp.
Ligation of dna material to be supplied by the user nuclease free water cat. Ligafasttm rapid dna ligation system product information. Pdf dna cloning without restriction enzyme and 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. Efficient synthesis of stably adenylated dna and rna. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. Both dna and rna ligases catalyze the formation of a phosphodiester bond between. 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.
Although the reactions catalyzed by the enzymes of e. Canonical nucleosides can be utilized by t4 dna ligase as. 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. The ligafast rapid dna ligation system is designed for the efficient ligation of stickyended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes.
Manual, second edition, cold spring harbor laboratory press, cold spring harbor. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. The enzyme will not join singlestranded nucleic acids. T4 dna ligase is unstable at temperatures over 30 c. There must be no visible nicking or cutting of the dna. Promega enzyme resource guide, cloning enzymes, br075b. The flexi vector system is a directional cloning method for proteincoding sequences. 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. Efficient synthesis of stably adenylated dna and rna adapters. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. 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. Following incubation, the supercoiled dna is visualized on an ethidium bromidestained agarose gel.
Ligafast rapid dna ligation system protocolpdf promega. We postulated that by switching to single stranded t4. T4 dna ligase is used in molecular cloning experiments to ligate join the. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. T4 dna ligase bluewhite cloning qualified protocol promega. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. This method takes advantage of the property of t4 dna ligase to join rna molecules when they are in an rna. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. Set up the following reaction in a microcentrifuge tube on ice. Learn more about our custom options for this product at. Do not heat inactivate if there is peg in the reaction buffer quick ligation kit buffer.
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