T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Ligation efficiency is also contingent on the integrity of the cohesive ends of the fragments being ligated. T4 dna ligase bluewhite cloning qualified protocol promega. There is considerable latitude in the temperature and time needed for successful ligations. 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.
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. Although the reactions catalyzed by the enzymes of e. The enzyme will not join singlestranded nucleic acids. 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. 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. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp. Efficient synthesis of stably adenylated dna and rna adapters.
The most commonly used is the t4 dna ligase method. Heat inactivate at 65c for 10 min or at 70c for 5 min. Following incubation, the supercoiled dna is visualized on an ethidium bromidestained agarose gel. 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. Ligafast rapid dna ligation system protocolpdf promega. L of ligation master mix to each well of a new pcr plate. Pdf dna cloning without restriction enzyme and ligase. Singlestranded nucleic acids are not substrates for. Ligation of dna material to be supplied by the user nuclease free water cat. T4 dna ligase ligase dna ligation promega corporation. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. L of purified pcr product from the pcr cleanup plate, 2. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp.
The flexi vector system is a directional cloning method for proteincoding sequences. Manual, second edition, cold spring harbor laboratory press, cold spring harbor. 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. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. 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. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. Efficient dna ligation in dnarna hybrid helices by. 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.
T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. 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 joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration. Learn more about our custom options for this product at. We postulated that by switching to single stranded t4.
Ligation protocol with t4 dna ligase m0202 protocols. Dna ligation with t4 dna ligase m0202 new england biolabs. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 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. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. 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 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. 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 unstable at temperatures over 30 c. The roche t4 dna ligase manual im using says ligation should be kept for 16h. Promega enzyme resource guide, cloning enzymes, br075b. 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.
Promega ligafast rapid dna ligation system 2x rapid ligation. Do not heat inactivate if there is peg in the reaction buffer quick ligation kit buffer. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. Canonical nucleosides can be utilized by t4 dna ligase as. Ligafasttm rapid dna ligation system product information. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Note that the table shows a ligation using a molar ratio of 1.
Bacteriophage t4 dna ligase is the ligase most commonly used in the. Singlestranded dna library preparation from highly degraded. 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. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. 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. Dna ligase is a specific type of enzyme, a ligase, ec 6. For this we utilized t4 dna ligase promega under the standard manufacturers conditions.
Both dna and rna ligases catalyze the formation of a phosphodiester bond between. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Set up the following reaction in a microcentrifuge tube on ice. 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. A pool of 60 nt acceptor oligonucleotides 60n were ligated to 10 pmol of a 3. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Dna cloning without restriction enzyme and ligase article pdf available in biotechniques 276. Ligation products were visualized on a 10% denaturing. 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.
Mix thoroughly, spin briefly and incubate for 1 hour at 22c. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. T4 dna ligase is provided with 10x reaction buffer. Oct 26, 2015 rna and dna adenylation by t4 rna ligase 1. Gently mix the reaction by pipetting up and down and microfuge briefly. Please remember to supplement the reaction with 1 mm atp final concentration. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1.
Singlestranded dna ligation with t4 dna ligase and circligase. Ligase from t4infected escherichia coli and promega t4 dna ligase buffer. Note t4 dna ligase is active in pcr and restriction. The optimal insert to vector dna ratio, is usually between 2. Promega ligafast rapid dna ligation system 2x rapid.
T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Efficient synthesis of stably adenylated dna and rna. 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. A previously reported method used double stranded t4 dna ligase to perform enzymatic adenylation 9. This method takes advantage of the property of t4 dna ligase to join rna molecules when they are in an rna. There must be no visible nicking or cutting of the dna. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases.
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