T4 dna ligase is provided with 10x reaction buffer. Binding of t4 dna ligase to dna may result in a band shift in agarose gels. It is related to the pol b family, which includes eukaryotic polymerases a, d, and e. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. 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. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. L of purified pcr product from the pcr cleanup plate, 2. Comparative analysis of t4 dna ligases and dna polymerases. In this study, we report results on the expression and purification of recombinant t4 dna ligase. Ligation protocol with t4 dna ligase m0202 protocols. Learn more about how this product is being used in the product citation tool.
It also joins dna fragments with either cohesive or blunt termini. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate. A similar structure, that of t7 dna ligase, has been solved subramanya et al. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. T4 dna ligase the enzyme efficiently joins blunt and. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Sequence alignment shows the two polymerases are 62% identical. T4 dna ligase from thermo fisher scientific selectscience. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. The enzyme will not join singlestranded nucleic acids. 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.
The structure of rb69 dna polymerase a 903 residue protein has been solved wang et al. 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. T4 dna ligase rapid the enzyme efficiently joins blunt. Using these buffers, the activity of t7 dna ligase is reduced approximately 10fold. Linear vector dna 20100 ng insert dna over vector thermo scientific 10x t4 dna ligase buffer 2. Bluntend ligation may be enhanced by addition of peg 4000 10% wv final. One unit catalyzes the exchange of 1 nmol 32plabeled pyrophosphate into atp in 20 min. Set up the following reaction in a microcentrifuge tube on ice. It is commonly used to join dna molecules to each other. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. T4 dna ligase is strongly inhibited by nacl or kcl if the concentration is 200mm.
One unit is equal to approximately 300 cohesiveend ligation units. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. Dnahind iii fragments analyzed by agarose gel electrophoresis. Adenylation of rna using t4 dna ligase, atp, and a complementary dna oligonucleotide that provides a recessed rna 5. 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. T4 dna ligase 200 units from thermo fisher scientific. L of ligase should be sufficient for larger ligation reactions. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. L reaction, scale the reaction size as necessary being sure to increase the amount of buffer proportionally. If the dna concentrations are low such that you cannot get all 100ng of dna, buffer and ligase into a 10.
L t4 dna ligase 5 u water, nucleasefree total volume 20. Dna ligase is a specific type of enzyme, a ligase, ec 6. 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. 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 structure reveals a prototypical atp. The t4 dna polymerase is an 898 amino acid residue protein. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well. The enzyme does not use atp for ligation but requires preadenylated linkers. T4 dna ligase will seal nicks for these dna substrates.
T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Dna insert ligation stickyend and bluntend into vector dna. 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 catalyzes the formation of a phosphodiester bond between the terminal 5 phosphate and a 3 hydroxyl groups of duplex dna or rna.
In order to obtain the maximum amount of activity from the ligase, a ph of 7. 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. L this protocol is for the dna insert ligation sticky end and bluntend into vector dna. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. 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 also joins dna fragments with either cohesive or blunt term. T4 dna ligase is the industry standard for performance and quality. Thermo scientific t4 dna ligase is a single polypeptide. Singlestranded nucleic acids are not substrates for thi. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. T4 dna ligase buffer from thermo fisher scientific. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1.
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