Get a printable copy pdf file of the complete article 882k. One unit is equal to approximately 300 cohesiveend ligation units. Ligation buffer for t4 dna ligase, 10 x conc 660 mm trishcl. T4 dna ligase has the ability to join bluntended dna fragments, and this activity is greatly enhanced by the addition of macromolecular crowding agents such as polyethylene glycol.
Mutation of k227 in t4 rna ligase 2 reduces enzyme. Its mw is about 62,000, and its optimal reaction ph is 7. For cohesive sticky ends, use 1 l of t4 dna ligase in a 20 l reaction for 10 minutes. Primary structure and genetic organization of phage t4 dna. The enzyme does not require atp for ligation but does need the preadenylated substrate. There is considerable latitude in the temperature and time needed for successful ligations. T4 rna ligase 2, truncated k227q t4 rnl2tr k227q specifically ligates the preadenylated 5. Dna ligases catalyze the formation of a phosphodiester bond between dna single. 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 joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration. 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. Dna ligase reconnects dna strands when they are broken. Ligation of bluntended and singlebase pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesiveend dna fragments.
T4 dna ligase catalyzes the formation of phosphodiester bonds between neighbouring 3hydroxyl and 5phosphate ends in doublestranded dna. Great for most common ligation applications including ta cloning. For details on nebs quality controls for dna ligases, visit our ligase quality page. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Commonly used nextgeneration sequencing ngs library preparation methods employ t4 dna ligase to join sequencerspecific adapters to the ends of. It has broder specificity and repairs single strended nicks in duplex dna, rna or dna. Protease inhibitor cocktail was purchased from roche indianapolis, in. Bluntend ligation may be enhanced by addition of peg 4000 10% wv final. T4 dna ligase competitor study nuclease contamination t4 dna ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3overhang or 5 overhang containing oligonucleotides.
T4 rna ligase 2, truncated kq t4 rnl2tr r55k, k227q specifically ligates the preadenylated 5. To thaw 5x t4 dna ligase at room temperature and vortex it vigorously to mix the components. T4 dna ligase rapid the enzyme efficiently joins blunt. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
T4 rna ligase 1 catalyses the formation of a phosphodiester bond between the terminal 5. 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. It is a monomeric polypeptide mw 68kda is encoded by bacteriophage gene30. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. Pdf cloning based on efficient threefragment assemby dna. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. 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. For convenience, ligations may be done at room temperature 2025c. Sitespecific mutagenesis experiments using t4 dna ligase or t4. T4 dna ligase rapid the enzyme efficiently joins blunt and. It also joins dna fragments with either cohesive or blunt term. A similar structure, that of t7 dna ligase, has been solved subramanya et al.
T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. T4 dna ligase is provided with 10x reaction buffer. Ligation of an oligo to single stranded dna cdna using t4 rna ligase 1m0204 overview. 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. The molecule has a mr of 55,230, and contains 487 amino acids. Singlestranded nucleic acids are not substrates for thi. Sweden, roche molecular biochemicals basel, switzer. It can be used to ligate cohesive or blunt end dna fragments. Dna ligase is a specific type of enzyme, a ligase, ec 6. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. T4 dna ligase is the industry standard for performance and quality. The primary structure of phage t4 dna ligase has been determined by dna sequencing of a cloned restriction fragment containing its gene, and partial amino acid sequence analysis of the protein. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications.
The ligation mixture contained 2 units of t4 dna ligase, 2 l of t4 dna ligase. Ligation of an oligo to single stranded dna cdna using. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. Singlestranded nucleic acids are not substrates for this enzyme. L of ligation master mix to each well of a new pcr plate. Construction of mtb72f plasmid as a dna vaccine candidate for.
Promega manufactures products for a number of intended uses. Please refer to the product label for the intended use statements for specific products. The aim of this study was to produce a dna vaccine of mtb72f. Learn more about how this product is being used in the product citation tool. Oct 07, 20 bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. Download a pdf containing pricing for our full product list. T4 rnl2tr r55k, k227q is a doublepoint mutant of t4 rna ligase 2. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5phosphate and 3hydroxyl ends in double strand dna or rna with blunt or cohesiveend. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i. Please remember to supplement the reaction with 1 mm atp final concentration. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Leveraging the unique knowhow of roche diagnostics and roche pharmaceuticals, we work with you to deliver high quality.
The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna 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. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5phosphate and 3hydroxyl ends in double strand dna or rna with blunt or cohesiveend termini. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Activity determination one unit of t4 dna ligase is defined as the amount of enzyme required to catalyze the ligation of greater than 90% of. Similar conditions are used for ligation anchored pcr 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. 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. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna, rna or dna rna hybrids. 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 rna ligase 1 t4 rna ligase 2 t4 rna ligase 2 truncated t4 rna ligase 2, truncated k227q t4 rna ligase 2, truncated kq thermostable 5 app dnarna ligase 5 adenylation kit rtcb ligase. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min.
Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. T4 dna ligase for t4 dna ligation, ta cloning, and other. One unit is defined as the amount of enzyme required to ligate 50% of an equimolar mix 125nm of a singlestranded 5. This enzyme will join bluntend and cohesiveend termini as well as repair single stranded nicks in duplex dna, rna, or dnarna hybrids. The enzyme does not use atp for ligation but requires preadenylated linkers. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. T4 dna ligase is supplied at either 2,000uul or,000uul along with 10x buffer. T4 dna ligase can be completely inactivated by a 10 min.
Please note that t4 dna ligase is extremely temperature sensitive and storage. Sample material dephosphorylated dna with either blunt or sticky ends. T4 dna ligase recombinant form of the enzyme from t4. Order saltt4 dna ligase close order splintr ligase. Pdf on jan 1, 1998, holm schneider and others published cloning based on efficient threefragment assemby dna. Pdf kinetics and thermodynamics of nick sealing by t4 dna ligase. Templateindependent ligation of singlestranded dna by t4. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. To spin the vial of canvax t4 dna ligase for a few seconds before pipetting the enzyme. The enzyme will not join singlestranded nucleic acids. Ligation of an oligo to single stranded dna cdna using t4. Note that the table shows a ligation using a molar ratio of 1.
Singlestranded nicks in doublestranded dna are also closed. For insertion of dna into plasmid vectors the vector. L of purified pcr product from the pcr cleanup plate, 2. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. 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. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. T4 rna ligase 2, truncated t4 rnl2tr specifically ligates the preadenylated 5. In contrast, atp is the cofactor for dna ligases from bacteriophages e.
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