T4 dna ligase is provided with 10x reaction 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 performance of this buffer depends on the integrity of the atp. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0.
For bacterial transformation we offer a choice of competent cells, including our unique single step krx strain. For other cloning applications we offer t4 dna kinase, calf alkaline phosphatase and other modifying enzymes. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. It also joins dna fragments with either cohesive or blunt term. T4 dna ligase is used in molecular cloning experiments to ligate join the. Minimum passing specification is dna repair, and recombination. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids.
For conventional cloning of dna fragments, we provide a variety of restriction enzymes and t4 dna ligase. T4 dna ligase bluewhite cloning qualified protocol promega. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. The genome of the pathogenic psychrophile aliivibrio salmonicida encodes one such putative atpdependent dna ligase 10. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. Learn more about t4 dna ligase 10x buffer, promega. If the dna is not ligated you may want to check the activity of your ligase by using lambda dna digested with hindiii cat. Straight lines indicate dna molecules, wavy lines indicate rna molecules and arrowheads. Incubate ligation reactions containing vector with 5 or 3 overhangs at 25c for 5 minutes. T4 rna ligase catalyzes the joining of singlestranded nucleic acids, including rnadna hybrids.
Ligation protocol with t4 dna ligase m0202 protocols. Set up the following reaction in a microcentrifuge tube on ice. For details on nebs quality controls for dna ligases, visit our ligase quality page. Pdf cloning and expression of a new bacteriophage shph. Activities of enzymes commonly used in cloning applications. 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. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector. Constructing genomic libraries using the pgemt vector. Promega enzyme resource guide 8 two ligases introduction dna ligases are primarily responsible for joining the gaps that form in dna during replication i. 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. Checking your t4 dna ligase activity thermo fisher.
The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Identification and remediation of nucleotide bias in ssdna ligation is crucial because such bias can fail to. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. 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. Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1.
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. 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. Sure cells increased stability of dna containing inverted repeats or zdna stratagene. Ligafasttm rapid dna ligation system product information. Use only the t4 dna ligase supplied with this system to perform pgem t and pgemt easy vector ligations. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dnarna hybrids. 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. 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 enzyme, purified from recombinant escherichia colica4 rnase i. The enzyme will not join singlestranded nucleic acids. 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.
Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. Recombinant expression and purification of an atpdependent. 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. Current ssdna ligation protocols use circligase i or t4 rna ligase and suffer from slow kinetics, poor yield and severe nucleotide preference. The best known rna ligase is bacteriophage t4 rna ligase. For ligation of dna fragments, 50ng about 25fmol of pgemt vector were mixed with fragments at a 1.
For this we utilized t4 dna ligase promega under the standard manufacturers conditions. A ligation control dna is included as a system check. 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. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. 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.
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. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. T4 dna ligase, 30 weiss ul 50 00 weiss u 10x t4 dna ligase buffer 5x1. Canonical nucleosides can be utilized by t4 dna ligase as. Page 17 safety data sheet according to 19072006ec, article 31. 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. M1804 t4 dna ligase, 500u 291,000 219,000 m8221 ligafast rapid dna ligation system, 30rxn 126,000 95,000 m8225 ligafast rapid dna ligationsystem, 150rxn 412,000 309,000. 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. Ligase enzymes catalyze the joining or ligation of dna or rna segments, where. This enzyme joins the break in the dna backbone structure by. The enzyme, purified from recombinant escherichia coli ca4 rnase ideficient, has an apparent molecular weight of 43. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. In order to study its structure and activity in vitro we have undertaken the recombinant production and puri.
In this study, we report results on the expression and purification of recombinant t4 dna ligase. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Each promega product is shipped with documentation. Use only promega t4 dna ligase supplied with this system to perform. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding.
The roche t4 dna ligase manual im using says ligation should be kept for 16h. This dna is highly pure and should be easily ligated using the protocol below. T4 dna ligase ligase dna ligation promega corporation. Cloning and expression of a new bacteriophage shph dna ligase isolated from sewage. 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. T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. T4 dna ligase catalyzes the joining of two strands of dna between the 5. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. As with t4 dna ligase, blunt end ligation is enhanced by the addition of peg 6000 to the reaction. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. The ligation products were collected with silica beads and dissolved in 10l te buffer. Incubate ligation reactions containing vector with blunt ends at 25c for 15 minutes. Promega enzyme resource guide, cloning enzymes, br075b. T4 dna ligase catalyzes the joining of two dna strands between the 5. T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto 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. We enable science by offering product choice, services, process excellence and our people make it happen. T4 dna ligase the enzyme efficiently joins blunt and.557 614 861 1006 1513 682 249 1314 958 1638 1089 802 1249 1562 922 474 532 605 1470 124 289 973 422 840 1669 909 782 423 1493 650 861 932 320 1040