T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm. Structural biochemistryt4 dna ligase wikibooks, open. Dna ligases play essential roles in dna replication and repair. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. Sequence alignment shows the two polymerases are 62% identical. The optimal temperature for a ligation is a balance between the optimal temperature for t4 dna ligase enzyme activity 16c and the temperature necessary to ensure annealing of the. Applications cloning of restriction enzyme generated dna fragments cloning of pcr products joining of doublestranded oligonucleotide linkers or adaptors to dna sitedirected mutagenesis amplified fragment length. Set up the following reaction in a microcentrifuge tube on ice. One unit catalyzes the exchange of 1 nmol 32plabeled pyrophosphate into atp in 20 min. Singlestranded nucleic acids are not substrates for this enzyme. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1.
T4 dna ligase is provided with 10x reaction buffer. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dna rna hybrids 1. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. T4 dna ligase the enzyme efficiently joins blunt and. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. T4 dna ligase structure reveals a prototypical atp. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. 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. The structure of rb69 dna polymerase a 903 residue protein has been solved wang et al. L of ligase should be sufficient for larger ligation reactions. 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. A similar structure, that of t7 dna ligase, has been solved subramanya et al.
T7 dna ligase is also active in buffers without peg 6000, such as our t4 dna ligase buffer and nebuffer 14, for applications in which peg 6000 is detrimental. It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well. Bacteriophage t4 dna ligase is the first atpdependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Linear vector dna 20100 ng insert dna over vector thermo scientific 10x t4 dna ligase buffer 2. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. L this protocol is for the dna insert ligation sticky end and bluntend into vector dna. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. T4 dna ligase from thermo fisher scientific selectscience. The t4 dna polymerase is an 898 amino acid residue protein.
It is related to the pol b family, which includes eukaryotic polymerases a, d, and e. It is commonly used to join dna molecules to each other. It also joins dna fragments with either cohesive or blunt term. Adenylation of rna using t4 dna ligase, atp, and a complementary dna oligonucleotide that provides a recessed rna 5. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Thermo scientific t4 dna ligase is a single polypeptide. Bluntend ligation may be enhanced by addition of peg 4000 10% wv final. T4 rna ligase 2, truncated kq t4 rnl2tr r55k, k227q specifically ligates the preadenylated 5. The structure of the t4 ligasedna complex was in turn determined by molecular replacement phasing using the structures of the t4 dna ligase nterminal domain and the chlorella virus ligasedna complex as search models and was refined to 2.
Ligation protocol with t4 dna ligase m0202 protocols. T4 dna ligase is the industry standard for performance and quality. T4 dna ligase 200 units from thermo fisher scientific. 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. There is considerable latitude in the temperature and time needed for successful ligations.
One unit is equal to approximately 300 cohesiveend ligation units. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. In order to obtain the maximum amount of activity from the ligase, a ph of 7. 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. Binding of t4 dna ligase to dna may result in a band shift in agarose gels. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. T4 dna ligase buffer from thermo fisher scientific. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. The enzyme repairs singlestrand nicks in duplex dna, rna or dna rna hybrids, joins dna fragments with either cohesive or blunt termini 1, 2. T4 dna ligase rapid the enzyme efficiently joins blunt. 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 buffer contains atp, so repeated freeze thaw cycles can degrade atp, thereby decreasing the efficiency of ligation. 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 a phosphodiester linkage between dna chains by condensing the 5phosphoryl group of one dna chain with the adjacent 3.
L reaction, scale the reaction size as necessary being sure to increase the amount of buffer proportionally. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. If the dna concentrations are low such that you cannot get all 100ng of dna, buffer and ligase into a 10. The ideal temperature for the t4 dna ligase reaction is 16 qc, however it works over a range of temperatures, so 4 qc or room temperature can also be used.
T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate. Dnahind iii fragments analyzed by agarose gel electrophoresis. T4 dna ligase will seal nicks for these dna substrates. Please remember to supplement the reaction with 1 mm atp final concentration.
The enzyme does not use atp for ligation but requires preadenylated linkers. Using these buffers, the activity of t7 dna ligase is reduced approximately 10fold. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. The enzyme will not join singlestranded nucleic acids. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna.
It also joins dna fragments with either cohesive or blunt termini. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. Singlestranded nucleic acids are not substrates for thi. Comparative analysis of t4 dna ligases and dna polymerases. Dna ligase is a specific type of enzyme, a ligase, ec 6. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. The most commonly used is the t4 dna ligase method. L t4 dna ligase 5 u water, nucleasefree total volume 20.
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