T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna strands with 3 hydroxyl and 5 phosphate termini in the presence of atp. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Great for most common ligation applications including ta cloning. For convenience, ligations may be done at room temperature 2025c. 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. Overall, the most active variants were p50ligase i. Sitedirected mutagenesis using pfu dna polymerase and t4. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. D2886 catalyzes the formation of a phosphodiester bond between the terminal 5.
Structural biochemistryt4 dna ligase wikibooks, open books. Templateaindependent ligation of singleastranded dna by t4. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. Schematic representation of the different t4 dna ligase mutants expressed in li as.
Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. 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. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. The enzyme efficiently joins blunt and cohesive ends and repairs singlestranded nicks in duplex dna, rna or dnarna hybrids. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. T4 dna ligase the enzyme efficiently joins blunt and cohesive.
Templateindependent ligation of singlestranded dna by t4. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and returned immediately to 20 c after use. The most commonly used is the t4 dna ligase method. Structural intermediates of a dnaligase complex illuminate. It is not appreciably active on bluntended substrates. Dna ligase is a specific type of enzyme, a ligase, ec 6. Please remember to supplement the reaction with 1 mm atp final concentration. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Pdf atpdependent dna ligases are essential enzymes in both dna replication and. Pdf t4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological. Engineered dna ligases with improved activities in vitro. 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.
Ligafasttm rapid dna ligation system certificate of. There is considerable latitude in the temperature and time needed for successful ligations. Pdf functional characterization of the t4 dna ligase. Singlestranded nucleic acids are not substrates for thi. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Sitedirected mutagenesis using pfu dna polymerase and t4 dna. Although the reactions catalyzed by the enzymes of e. 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. 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. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor.
This master mix is specifically formulated to improve ligation and transformation of both bluntend and singlebase overhang substrates. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. Dna ligase iii lig3 is the sole dna ligase in mitochondria and as such is essential for all mitochondrial dna repair 26, 27. Consistent with this role, lig3 displays robust activity toward both single and. This master mix is specifically formulated to improve ligation and. B p50 fused to t4 dna ligase and ligasectf t4 dna ligase fused to an artificial, chimeric transcription factor. 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. On the basis of an analogous approach to dna 59adenylation using t4 dna ligase chiuman and li 2002 and our own procedure for rna 59adenylation with t4 rna ligase silverman 2004, here we. One microliter approximately 100 ng of each pcr product was directly used for bluntend ligation 1 h at room temperature, 1.
Most commonly, the protein enzymes t4 dna ligase or t4 rna ligase are used to activate the 5 0terminal phosphate of the donor rna fragment by adenylation and join it to the 3 0hydroxyl group of. Atp is not stable and decreased concentrations of atp largely influence the ligation efficiency. L of ligation master mix to each well of a new pcr plate. L of purified pcr product from the pcr cleanup plate, 2. Bamhi site of the ptrchisa expression vector invitrogen. The level of t4 dna ligase expression was monitored by western blotting.
Ligation protocol with t4 dna ligase m0202 protocols. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. One unit is equal to approximately 300 cohesiveend ligation units. 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. For cohesive sticky ends, use 1 l of t4 dna ligase in a 20 l reaction for 10 minutes. Bluntta ligase master mix is a readytouse solution of t4 dna ligase, proprietary ligation enhancer, and optimized reaction buffer. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications. The role of divalent metal ions in enzymatic dna ligation. Structural biochemistryt4 dna ligase wikibooks, open.
In order to obtain the maximum amount of activity from the ligase, a ph of 7. Here we report that this ligase, unlike escherichia coli dna ligase, taq dna ligase and. Singlestranded nucleic acids are not substrates for this enzyme. T4 dna ligase for t4 dna ligation, ta cloning, and other. T4 dna ligase rapid the enzyme efficiently joins blunt. Enzymatic manipulation of nucleic acidligation protocols. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Dna ligase reconnects dna strands when they are broken. The enzyme will not join singlestranded nucleic acids. T4 dna ligase catalyzes the joining of two strands of dna between the 5. Dna ligation practical approach online, oxford university press ligation reaction using t4 ligase. T4 dna ligase is provided with 10x reaction buffer. Set up the following reaction in a microcentrifuge tube on ice.
It can be used to ligate cohesive or blunt end dna fragments. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Purification followed the same scheme as for pmarlig and amelig, with the exception that tag removal was omitted and single round of imac was performed prior to gel filtration. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master mix. T4 dna ligase rapid the enzyme efficiently joins blunt and. Pdf templateindependent ligation of singlestranded dna by t4. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. Here we report that this ligase, unlike escherichia coli dna ligase, taq dna ligase and ampligase, is able to join the ends of singlestranded dna in the absence of any duplex dna structure at the ligation site. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna.
932 1457 1088 281 1264 71 430 77 250 658 1045 1183 685 1211 946 266 399 1185 1045 782 969 660 1199 155 246 164 996 602 1406 791 753 457 2 983 1372 751 870 851 229 400 1320 828 569