Both companies tech support provided inconclusive answers. The ng of insert can be obtained by using the below equation. 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 rapid the enzyme efficiently joins blunt and. Pdf on jul 1, 2017, luigi marongiu and others published ligationmediated pcr find, read and cite all the research you need on researchgate. Ligafasttm rapid dna ligation system product information. 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. 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.
Set up the following reaction in a microcentrifuge tube on ice. A protocol for combining two strands of dna between the 5. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Singlestranded nucleic acids are not substrates for this enzyme.
T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. Manual, second edition, cold spring harbor laboratory press, cold spring harbor. T7 dna ligase is also active in buffers without peg 6000, such as our t4 dna ligase buffer and nebuffer 14, for applications in which peg 6000 is detrimental. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Ive heard that both reagents can be substituted with promegas t4 dna ligase and buffer attached file to yield highquality results. 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. T4 dna ligase catalyzes the joining of two strands of dna between the 5. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna.
Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. Vector dna 100 ng insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Dna ligase is a specific type of enzyme, a ligase, ec 6. The insert dna can easily be removed for subcloning using an ecori single digest. The enzyme will not join singlestranded nucleic acids. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. Promega ligafast rapid dna ligation system 2x rapid. T4 dna ligase for t4 dna ligation, ta cloning, and other. The insert dna can easily be removed for subcloning using an. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. Pathway complexity in fueldriven dna nanostructures with.
Ligation efficiency is also contingent on the integrity of the cohesive ends of the fragments being ligated. Singlestranded dna ligation with t4 dna ligase and circligase. Please remember to supplement the reaction with 1 mm atp final concentration. Ligafast rapid dna ligation system protocolpdf promega. Once you thaw a tube with buffer discard, do not reuse or refreeze. Ligafast rapid dna ligation system protocol promega. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. 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 the enzyme efficiently joins blunt and cohesive. I have tried promega t4 ligase and invitrogen t4 ligase side by side with the same dna and found the promega ligase works better. Because the ligase buffer contains atp, if using ligase for the first time aliquot out buffer into 10 20. Minimum passing specification is t4 dna ligase contd. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7.
T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. There must be no visible nicking or cutting of the dna. T4 dna ligase ligase dna ligation promega corporation. Restriction digest, dephosphorylation, gel purification. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences.
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. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja. Promega manufactures products for a number of intended uses. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. L of ligase should be sufficient for larger ligation reactions. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. 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. The optimal insert to vector dna ratio, is usually between 2. A pool of 60 nt acceptor oligonucleotides 60n were ligated to 10 pmol of a 3. A similar structure, that of t7 dna ligase, has been solved subramanya et al. T4 dna ligase is provided with 10x reaction buffer.
T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Singlestranded dna library preparation from highly degraded. 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 best known rna ligase is bacteriophage t4 rna ligase. L of purified pcr product from the pcr cleanup plate, 2. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Learn more about how this product is being used in the product citation tool. Ligation protocol with t4 dna ligase m0202 protocols. T4 dna ligase is unstable at temperatures over 30 c. Following incubation, the supercoiled dna is visualized on an ethidium bromidestained agarose gel. Recombinant clones can be efficiently selected using bluewhite screening on indicator plates. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful.
The performance of this buffer depends on the integrity of the atp. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. T4 dna ligase bluewhite cloning qualified protocol promega. 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. Test of bsmbi and t7 dna ligase procedure using neb buffer 3. Promega enzyme resource guide, cloning enzymes, br075b. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. T4 dna ligase rapid the enzyme efficiently joins blunt. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english.
It also joins dna fragments with either cohesive or blunt term. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. 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. T4 dna ligase, 30 weiss ul 50 00 weiss u 10x t4 dna ligase buffer 5x1. Higher concentrations of dna reaction components will result in a higher rate of reaction.
L of ligation master mix to each well of a new pcr plate. The pt arge t mammalian expression vector system includes jm109 high efficiency competent cells for transformation of ligation reactions. L aliquots of the reaction solution were collected and quenched by 4. Restriction digest, dephosphorylation, gel purification, ligation. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of. 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. Promega ligafast rapid dna ligation system 2x rapid ligation. L reaction, scale the reaction size as necessary being sure to increase the amount of buffer proportionally. Promega vs neb t4 dna ligase as well as ligase buffer. Using these buffers, the activity of t7 dna ligase is reduced approximately 10fold. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. 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.