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. What problems can be encountered in the restriction digest that can cause ligation using t4 dna ligase or subsequent transformation to fail. T4 dna ligase t4 dna ligase t4 bacteriophage of escherichia coli high efficient t4 dna ligase, joins more than 95 % of dna fragments within less than 1 h. Purification of the t4 dna ligase by blue sepharose. 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. In molecular biology it is commonly used for the insertion of restriction enzyme. It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well. Singlestranded nucleic acids are not substrates for this enzyme.
Titration of p50 t4 dna ligase prior to a ligation attempt. Temperature optimum of the most commonly using t4 dna ligase is around 37. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence. Thaw the t4 dna ligase buffer and resuspended at room temperature. In order to obtain the maximum amount of activity from the ligase, a ph of 7. The mechanism of the ligation reaction was first elucidated in the laboratory of i. Fastdigest restriction enzymesthermo scientific us. T4 dna ligase is provided with 10x reaction buffer. D2886 catalyzes the formation of a phosphodiester bond between the terminal 5. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
Heat inactivate at 65c for 10 min or at 70c for 5 min. Singlestranded nucleic acids are not substrates for. Ligation protocol with t4 dna ligase m0202 protocols. Similarly to mammalian dna ligase i, t4 dna ligase can relax supercoiled dna in an ampdependent reaction that could be referred to as the reverse reaction 7,8. Unit definition one weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmole of p32 from pyrophosphate into. L of ligase should be sufficient for larger ligation reactions. This protocol is for the selfcircularization of linear dna.
The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. For rapid ligation of blunt ends, use t4 dna ligase, cat no. It has been shown that t4 dna ligase seals dsdna substrates containing an abasic site or a gap at the ligation junction, joins branched dna. By joining the 3hydroxy and 5phosphate termini to form a phosphodiester, dna ligases are absolutely essential for dna replication and repair in all organisms. Size e106001 20 000 ce units 300 weiss units e106002 100 000 ce units 1500 weiss. T4 rna ligase is a different enzyme than t4 dna ligase. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master mix. Two fragments of dna may be joined together by dna ligase which catalyzes the formation of a phosphodiester bond between the 3oh at one end of a strand of dna and the 5phosphate group of another. I used 16c overnight and 20c 2h, there was no difference with neb t4 ligase and a fermentas kit, where 5 minutes at 22c was suggested. Structural biochemistryt4 dna ligase wikibooks, open books. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. 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. 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.
Note t4 dna ligase is active in pcr and restriction. The enzyme efficiently joins blunt and cohesive ends and repairs singlestranded nicks in duplex dna, rna or dna rna hybrids. 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 5x rapid ligation buffer water, nucleasefree detailed protocol note prior to electroporation, it is necessary to inactivate t4 dna ligase by chloroform extraction or spin column purification, e. Ligation protocol with t4 dna ligase m0202 updated 2842019 3. The rna ligase should be able to ligate oligos to ssdna. In animals and bacteriophage, atp is used as the energy source for the. T4 dna ligase catalyzes the joining of two strands of dna between the 5. The rapid dna ligation kit is functionally tested in cloning experiment. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. Analytical biochemistry 108, 227229 1980 purification of the t4 dna ligase by blue sepharose chromatography masahiro sugiura national institute of genetics, mishima, shizuokaken 411, japan received march 31, 1980 t4 dna ligase catalyzes the formation of phosphodiester bonds between adjacent 5phosphoryl and 3hydroxyl ends in nicked duplex dna 1. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Than i perform ligation with both fermentas t4 dna ligase and neb t4 dna ligase, i also.
The 5 end of the fragment needs to be phosphorylated for ligation to occur that is the end of the fragment participating in the ligation site. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dnarna hybrids 1. 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 the terminal 5 phosphate and a 3 hydroxyl groups of duplex dna or rna. It also joins dna fragments with either cohesive or blunt term. Singlestranded nucleic acids are not substrates for thi. Sitedirected mutagenesis using pfu dna polymerase and t4 dna. Ligation protocol with t4 dna ligase m0202 benchling. Higher concentrations of dna reaction components will result in a higher rate of reaction. The protein encoded by this gene is a dna ligase that joins singlestrand breaks in a doublestranded polydeoxynucleotide in an atpdependent reaction. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Therefore, enzymes used in downstream applications can be directly added to the fastdigest reaction mix. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. 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.
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. Ligation efficiency is also contingent on the integrity of the cohesive ends of the fragments being ligated. It covalently joins the phosphate backbone of dna with blunt or compatible cohesive ends see figure 1 and its natural role is in repairing double strand breaks in dna molecules. Covalently joins sticky and blunt ended dsdna fragments and seals singlestranded nicks. Dna rna modifying enzymes, such as ligases, phosphatases, kinases and mesophilic dna polymerases have 100% activity in fastdigest and fastdigest green buffer. For details on nebs quality controls for dna ligases, visit our ligase quality page. 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 most commonly used is the t4 dna ligase method. Here, we describe a general protocol for the use of goldbio t4 dna ligase to ligate vector and insert dna. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp. The enzyme repairs singlestrand nicks in duplex dna, rna or dnarna hybrids but has no activity on singlestranded nucleic acids. Ligafasttm rapid dna ligation system certificate of. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5.
Rapid ligation protocol for plasmid cloning of dna fragments. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. T4 dna ligase is supplied in a solution containing 10 mm trishcl ph 7. T4 dna ligase is an atpdependent ligase that catalyzes a joining reaction between dna molecules. L reaction, scale the reaction size as necessary being sure to increase the amount of buffer proportionally. Dna ligase is a specific type of enzyme, a ligase, ec 6. One t4 dna ligase cohesive end unit is equivalent to approximately 3 cohesive end units as measured with a lambdahind iii dna fragment substrate in 1x t4 dna ligase reaction buffer. Reaction setup component, volume l, final concentration.
The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna ligation. One weiss unit is approximately equivalent to 22 units. Review and cite dna ligase protocol, troubleshooting and other. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Page 3 of 4 rapid ligation of cohesive ends 5min for plasmid cloning of dna fragments. 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. In order to maximize transformation efficiency of the correct insertvector combination, the protocol provided is recommended.
Although the reactions catalyzed by the enzymes of e. The following protocol is for rapid ligation of cohesive ends. Therefore, the best choice for bluntended dna fragments is 37. Dna ligase from t4infected escherichia coli buffered. The optimal insert to vector dna ratio, is usually between 2. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. 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. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and. If the dna concentrations are low such that you cannot get all 100ng of dna, buffer and ligase into a 10. The enzyme will not join singlestranded nucleic acids.
It is recommended that the reaction buffer be discarded after one year of storage at 20 c and replaced with fresh buffer to ensure maximum performance. What controls should be run to test the cells and dna when using t4 dna ligase. T4 dna ligase can ligate other substrates with reduced efficiency, including. The enzyme repairs singlestrand nicks in duplex dna, rna or dna rna hybrids but has no activity on singlestranded nucleic acids. One microliter approximately 100 ng of each pcr product was directly used for bluntend ligation 1 h at room temperature, 1. Rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. T4 dna ligase buffer contains atp, so repeated freeze thaw cycles can degrade atp, thereby decreasing the efficiency of ligation. Cloning systema complete, onebuffer, oneprotocol system for beautifully.
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 ends. Thermo scientific rapid dna ligation kit for 150 reactions. Sitedirected mutagenesis using pfu dna polymerase and t4. Mix thoroughly, spin briefly and incubate for 1 hour at 22c. Templateindependent ligation of singlestranded dna by t4.
Fast ligation efficiency is equal to that obtained with t4 dna ligase in a standard 1 hour ligation. Please see the neb website for supporting information on this protocol. Set up the following reaction in a microcentrifuge tube on ice. T4 dna ligase rapid the enzyme efficiently joins blunt and. 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 catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. T4 dna ligase is inhibited by metal chelators, phosphate and ammonium ions, kcl and. From the kinetics of the reaction and from the absence of nicked dna in the final products it was inferred that dna ligase acts processively through a nickingclosing mechanism. I chose to use 98 nm concentration and had success after transforming with the above protocol 10min at rt in neb t4 ligase buffer, 65c15min heat inactivation, transformed 1 ul into dh5alpha e. Note that the table shows a ligation using a molar ratio of 1.
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