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. T4 dna ligase recombinant form of the enzyme from t4 phage. Singlestranded nicks in doublestranded dna are also closed by t4 dna ligase. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Oct 07, 20 bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. T4 dna ligase for t4 dna ligation, ta cloning, and other.
However, purified t4 dna ligase obtained from bacteria that contain cloned t4 gene 30 dna catalyzes bluntend joining. Set up the following reaction in a microcentrifuge tube on ice. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. The hisk 159 l mutant bears a substitution of lys159 in the active site site i.
Bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. Singlestranded nucleic acids are not substrates for this enzyme. 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 recombinant form of the enzyme from t4. To produce the three deletion mutants we used phis t4 as template in a series of pcr reactions. Kinetics and thermodynamics of nick sealing by t4 dna ligase.
One unit is defined as the amount of enzyme required to ligate 50% of an equimolar mix 125nm of a singlestranded 5. Structural biochemistryt4 dna ligase wikibooks, open books. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. The molecule has a mr of 55,230, and contains 487 amino acids. Schematic representation of the different t4 dna ligase mutants expressed in li as histagged proteins a.
The most commonly used is the t4 dna ligase method. T4 rna ligase 1 catalyzes the ligation of a 5 phosphorylterminated nucleic acid donor to a 3 hydroxylterminated nucleic acid acceptor through the formation of a 3. T4 dna ligase is provided with 10x reaction buffer. It has broder specificity and repairs single strended nicks in duplex dna, rna or dna. Structural biochemistryt4 dna ligase wikibooks, open. Please remember to supplement the reaction with 1 mm atp final concentration. A similar structure, that of t7 dna ligase, has been solved subramanya et al. One weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1. Pdf on jan 1, 1998, holm schneider and others published cloning based on efficient threefragment assemby dna. T4 rna ligase 1 catalyses the formation of a phosphodiester bond between the terminal 5. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and. In order to obtain the maximum amount of activity from the ligase, a ph of 7. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and returned immediately to 20 c after use. Oct 25, 1983 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.
For cohesive sticky ends, use 1l of t4 dna ligase in a 20l reaction for 10 minutes. For blunt ends, use 1l of t4 dna ligase in a 20l reaction for 2 hours. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min. L roche molecular biochemicals, indianapolis, in see note 5. Great for most common ligation applications including ta cloning. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. Ligation protocol with t4 dna ligase m0202 protocols. L of purified pcr product from the pcr cleanup plate, 2. Ligation buffer for t4 dna ligase, 10 x conc 660 mm trishcl.
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. Dna ligase is a specific type of enzyme, a ligase, ec 6. 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. 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. There is considerable latitude in the temperature and time needed for successful ligations. Singlestranded nucleic acids are not substrates for thi. For convenience, ligations may be done at room temperature 2025oc.
T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. This method takes advantage of the property of t4 dna ligase to join rna molecules when they are in an rna. Primary structure and genetic organization of phage t4 dna. 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 phosphodi ester bonds. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna using atp as a cofactor. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. The enzyme will not join singlestranded nucleic acids.
The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. Singlestranded nicks in doublestranded dna are also closed. T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm. Learn more about how this product is being used in the product citation tool. Application ligation of sticky and bluntended dna fragments. Contents t4 dna ligase, supplied with 10x concentrated ligation buffer that includes atp. T4 dna ligase can be used to join dna fragments with staggered or blunt ends.
T4 rna ligase catalyzes the circularization of homopolyribonucleotides with a 3. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in. It is a monomeric polypeptide mw 68kda is encoded by bacteriophage gene30. 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 neighbouring 3hydroxyl and 5phosphate ends in.
Oct 16, 2003 t4 dna ligase is an enzyme that catalyses formation of the phosphodiester bond between the adjacent 5po 4 and 3oh groups of two dsdna fragments. 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. 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. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. L of ligation master mix to each well of a new pcr plate. The position of the conserved boxes see text is indicated. Its mw is about 62,000, and its optimal reaction ph is 7. It also carries out intermolecular reactions with either rna or 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. The level of t4 dna ligase expression was monitored by western blotting using anti t4 dna ligase antibodies. T4 dna ligase is the industry standard for performance and quality. For dilution of the enzyme roche recommends using a buffer containing the components of the storage buffer. The observation that rna ligase stimulates the bluntend joining reaction of dna ligase by increasing the v, of the reaction suggests that these two enzymes might interact in, vivo 51.
It can be used to ligate cohesive or blunt end dna fragments. Bluntend ligation may be enhanced by addition of peg 4000 10% wv final. A clone phis t4 was selected and completely sequenced sequenase version ii. Dnahind iii fragments analyzed by agarose gel electrophoresis. One unit is defined as the amount of enzyme required to give 50% ligation of hind iii fragments of dna 5. Although the reactions catalyzed by the enzymes of e. The enzyme can be inactivated by heating 65 c, 10 minutes.
In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. T4 dna ligase buffer contains atp, so repeated freeze thaw cycles can degrade atp, thereby decreasing the efficiency of ligation. It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well. T4 dna ligase can be used to join dna fragments with staggered or blunt ends and to repair nicks in doublestranded dna having 3hydroxyl and 5phosphate.