L of purified pcr product from the pcr cleanup plate, 2. Bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. T4 dna ligase recombinant form of the enzyme from t4 phage. Application ligation of sticky and bluntended dna fragments. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. Structural biochemistryt4 dna ligase wikibooks, open books. It has broder specificity and repairs single strended nicks in duplex dna, rna or dna. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. One unit is defined as the amount of enzyme required to give 50% ligation of hind iii fragments of dna 5. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. 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 buffer contains atp, so repeated freeze thaw cycles can degrade atp, thereby decreasing the efficiency of ligation. Kinetics and thermodynamics of nick sealing by t4 dna ligase. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and. There is considerable latitude in the temperature and time needed for successful ligations. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. 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. The molecule has a mr of 55,230, and contains 487 amino acids. 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. It is a monomeric polypeptide mw 68kda is encoded by bacteriophage gene30. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. The position of the conserved boxes see text is indicated.
One weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1. One unit is defined as the amount of enzyme required to ligate 50% of an equimolar mix 125nm of a singlestranded 5. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. Structural biochemistryt4 dna ligase wikibooks, open. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and returned immediately to 20 c after use. Set up the following reaction in a microcentrifuge tube on ice.
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 most commonly used is the t4 dna ligase method. T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm. Schematic representation of the different t4 dna ligase mutants expressed in li as histagged proteins a. Contents t4 dna ligase, supplied with 10x concentrated ligation buffer that includes atp. T4 dna ligase catalyzes the formation of phosphodiester bonds between neighbouring 3hydroxyl and 5phosphate ends in. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Although the reactions catalyzed by the enzymes of e.
Bluntend ligation may be enhanced by addition of peg 4000 10% wv final. However, purified t4 dna ligase obtained from bacteria that contain cloned t4 gene 30 dna catalyzes bluntend joining. 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. Great for most common ligation applications including ta cloning. T4 rna ligase catalyzes the circularization of homopolyribonucleotides with a 3.
The level of t4 dna ligase expression was monitored by western blotting using anti t4 dna ligase antibodies. T4 dna ligase for t4 dna ligation, ta cloning, and other. Primary structure and genetic organization of phage t4 dna. To produce the three deletion mutants we used phis t4 as template in a series of pcr reactions. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Singlestranded nucleic acids are not substrates for this enzyme. 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.
For cohesive sticky ends, use 1l of t4 dna ligase in a 20l reaction for 10 minutes. One unit is equal to approximately 300 cohesiveend ligation units. Sample material dephosphorylated dna with either blunt or sticky ends. Its mw is about 62,000, and its optimal reaction ph is 7. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in. For convenience, ligations may be done at room temperature 2025oc. The hisk 159 l mutant bears a substitution of lys159 in the active site site i. For dilution of the enzyme roche recommends using a buffer containing the components of the storage buffer. Oct 07, 20 bacteriophage t4 dna ligase atp the most widely used dna ligase is derived from the t4 bacteriophage. 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. T4 dna ligase is the industry standard for performance and quality. Singlestranded nucleic acids are not substrates for thi. A similar structure, that of t7 dna ligase, has been solved subramanya et al. It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well.
T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. 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. Singlestranded nicks in doublestranded dna are also closed. T4 rna ligase 1 catalyses the formation of a phosphodiester bond between the terminal 5. L roche molecular biochemicals, indianapolis, in see note 5. Ligation protocol with t4 dna ligase m0202 protocols. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. 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. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. Singlestranded nicks in doublestranded dna are also closed by t4 dna ligase. 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. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. Learn more about how this product is being used in the product citation tool.
L of ligation master mix to each well of a new pcr plate. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. This method takes advantage of the property of t4 dna ligase to join rna molecules when they are in an rna. The enzyme can be inactivated by heating 65 c, 10 minutes. 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. T4 dna ligase is provided with 10x reaction buffer. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp. Pdf cloning based on efficient threefragment assemby dna. Dnahind iii fragments analyzed by agarose gel electrophoresis.
In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes. 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. A clone phis t4 was selected and completely sequenced sequenase version ii. For blunt ends, use 1l of t4 dna ligase in a 20l reaction for 2 hours. 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. In order to obtain the maximum amount of activity from the ligase, a ph of 7. T4 dna ligase recombinant form of the enzyme from t4. The enzyme will not join singlestranded nucleic acids. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes.
It also carries out intermolecular reactions with either rna or dna. 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. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min.