For this we utilized t4 dna ligase promega under the standard manufacturers conditions. In this study, we report results on the expression and purification of recombinant t4 dna ligase. Pdf cloning and expression of a new bacteriophage shph. T4 dna ligase catalyzes the joining of two strands of dna between the 5. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in,biological,biology supply,biology supplies,biology product.
Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. 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. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Ligase enzymes catalyze the joining or ligation of dna or rna segments, where. As with t4 dna ligase, blunt end ligation is enhanced by the addition of peg 6000 to the reaction. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. In order to study its structure and activity in vitro we have undertaken the recombinant production and puri.
For ligation of dna fragments, 50ng about 25fmol of pgemt vector were mixed with fragments at a 1. 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 nucleic acids. T4 dna ligase can catalyze the joining of rna to either a dna or rna strand in a duplex molecule but will not join singlestranded nucleic acids. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. T4 dna ligase catalyzes the joining of two dna strands between the 5. 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.
For details on nebs quality controls for dna ligases, visit our ligase quality page. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. Ligafasttm rapid dna ligation system product information. A ligation control dna is included as a system check. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Fiveminute incubations at 25c are used for kpni and ecor i, and 15 minute incubations at. Dna oligonucleotide to ligate to an unknown 3end of a cdna. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. The enzyme, purified from recombinant escherichia coli ca4 rnase ideficient, has an apparent molecular weight of 43. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. T3 dna ligase exhibits a higher tolerance 2fold for nacl in the reaction compared to t4 dna ligase, making the enzyme a versatile choice for in vitro molecular biology protocols requiring dna ligase activity. Recombinant expression and purification of an atpdependent. The genome of the pathogenic psychrophile aliivibrio salmonicida encodes one such putative atpdependent dna ligase 10. Checking your t4 dna ligase activity thermo fisher.
The ligafast rapid dna ligation system is designed for the efficient ligation of stickyended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes. For use with application, joining doublestranded dna molecules with cohesive or blunt ends. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. T4 dna ligase ligase dna ligation promega corporation. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences.
Sure cells increased stability of dna containing inverted repeats or zdna stratagene. 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. It also joins dna fragments with either cohesive or blunt term. Incubate ligation reactions containing vector with blunt ends at 25c for 15 minutes. Activities of enzymes commonly used in cloning applications. Straight lines indicate dna molecules, wavy lines indicate rna molecules and arrowheads. The enzyme, purified from recombinant escherichia colica4 rnase i. 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 nucleic. Constructing genomic libraries using the pgemt vector.
Promega corporation 2800 woods hollow road madison, wi 537115399 usa. Cloning and expression of a new bacteriophage shph dna ligase isolated from sewage. T4 dna ligase is provided with 10x reaction buffer. This enzyme joins the break in the dna backbone structure by. Current ssdna ligation protocols use circligase i or t4 rna ligase and suffer from slow kinetics, poor yield and severe nucleotide preference. T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5. We enable science by offering product choice, services, process excellence and our people make it happen. Page 17 safety data sheet according to 19072006ec, article 31. Product has been classified in accordance with the hazard criteria of the cpr and the msds contains all of the information required by the cpr. The ligation products were collected with silica beads and dissolved in 10l te buffer. Set up the following reaction in a microcentrifuge tube on ice. Promega enzyme resource guide, cloning enzymes, br075b. Each promega product is shipped with documentation. Use only the t4 dna ligase supplied with this system to perform pgem t and pgemt easy vector ligations.
Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. T4 rna ligase catalyzes the joining of singlestranded nucleic acids, including rnadna hybrids. Canonical nucleosides can be utilized by t4 dna ligase as. This kit contains t4 dna ligase, the enzyme of choice for virtually all cloning purposes because of its ability to ligate both cohesive and bluntended strands of dna. 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. If the dna is not ligated you may want to check the activity of your ligase by using lambda dna digested with hindiii cat. T4 dna ligase is used in molecular cloning experiments to ligate join the. Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector. This dna is highly pure and should be easily ligated using the protocol below.
Use only promega t4 dna ligase supplied with this system to perform. T4 dna ligase buffer pack from promega description t4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration. 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.
Incubate the reaction at room temperature for 3 hours, or at 4c overnight. For other cloning applications we offer t4 dna kinase, calf alkaline phosphatase and other modifying enzymes. For bacterial transformation we offer a choice of competent cells, including our unique single step krx strain. Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. 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. Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. The best known rna ligase is bacteriophage t4 rna ligase.
The performance of this buffer depends on the integrity of the atp. Incubate ligation reactions containing vector with 5 or 3 overhangs at 25c for 5 minutes. For conventional cloning of dna fragments, we provide a variety of restriction enzymes and t4 dna ligase. Identification and remediation of nucleotide bias in ssdna ligation is crucial because such bias can fail to. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dnarna hybrids. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Minimum passing specification is dna repair, and recombination. T4 dna ligase, 30 weiss ul 50 00 weiss u 10x t4 dna ligase buffer 5x1.
Learn more about t4 dna ligase 10x buffer, promega. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a cohesiveended or bluntended configuration. Ligation protocol with t4 dna ligase m0202 protocols. The roche t4 dna ligase manual im using says ligation should be kept for 16h. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. This enzyme does not appear to have any role in nucleic acid metabolism in bacteriophage t4 infected li, but instead appears to be required for the attachment of the bacteriophages tail. Promega enzyme resource guide 8 two ligases introduction dna ligases are primarily responsible for joining the gaps that form in dna during replication i.
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