Promega t4 dna ligase pdf files

For use with application, joining doublestranded dna molecules with cohesive or blunt ends. T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5. Checking your t4 dna ligase activity thermo fisher. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. 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. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. T4 dna ligase is provided with 10x reaction buffer. The best known rna ligase is bacteriophage t4 rna ligase. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dnarna hybrids.

Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3. 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. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. The enzyme, purified from recombinant escherichia coli ca4 rnase ideficient, has an apparent molecular weight of 43. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Canonical nucleosides can be utilized by t4 dna ligase as. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Ligase enzymes catalyze the joining or ligation of dna or rna segments, where. 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. Promega enzyme resource guide 8 two ligases introduction dna ligases are primarily responsible for joining the gaps that form in dna during replication i. If the dna is not ligated you may want to check the activity of your ligase by using lambda dna digested with hindiii cat. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i.

Recombinant expression and purification of an atpdependent. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. Use only promega t4 dna ligase supplied with this system to perform. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. For conventional cloning of dna fragments, we provide a variety of restriction enzymes and t4 dna ligase.

A ligation control dna is included as a system check. T4 rna ligase catalyzes the joining of singlestranded nucleic acids, including rnadna hybrids. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. Straight lines indicate dna molecules, wavy lines indicate rna molecules and arrowheads.

T4 dna ligase catalyzes the joining of two strands of dna between the 5. In this study, we report results on the expression and purification of recombinant t4 dna ligase. The performance of this buffer depends on the integrity of the atp. Constructing genomic libraries using the pgemt vector. For ligation of dna fragments, 50ng about 25fmol of pgemt vector were mixed with fragments at a 1. For bacterial transformation we offer a choice of competent cells, including our unique single step krx strain. Current ssdna ligation protocols use circligase i or t4 rna ligase and suffer from slow kinetics, poor yield and severe nucleotide preference. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. 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. The enzyme will not join singlestranded nucleic acids. Fiveminute incubations at 25c are used for kpni and ecor i, and 15 minute incubations at.

T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. 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. For details on nebs quality controls for dna ligases, visit our ligase quality page. 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. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. Ligation protocol with t4 dna ligase m0202 protocols.

The ligation products were collected with silica beads and dissolved in 10l te buffer. 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. For this we utilized t4 dna ligase promega under the standard manufacturers conditions. We enable science by offering product choice, services, process excellence and our people make it happen. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. Ligafasttm rapid dna ligation system product information. Promega enzyme resource guide, cloning enzymes, br075b. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Pdf cloning and expression of a new bacteriophage shph.

This dna is highly pure and should be easily ligated using the protocol below. For other cloning applications we offer t4 dna kinase, calf alkaline phosphatase and other modifying enzymes. Sure cells increased stability of dna containing inverted repeats or zdna stratagene. Page 17 safety data sheet according to 19072006ec, article 31. Cloning and expression of a new bacteriophage shph dna ligase isolated from sewage. Learn more about t4 dna ligase 10x buffer, promega. 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. In order to study its structure and activity in vitro we have undertaken the recombinant production and puri. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. T4 dna ligase ligase dna ligation promega corporation. 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 repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. 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.

T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. T4 dna ligase, 30 weiss ul 50 00 weiss u 10x t4 dna ligase buffer 5x1. Incubate ligation reactions containing vector with 5 or 3 overhangs at 25c for 5 minutes. The genome of the pathogenic psychrophile aliivibrio salmonicida encodes one such putative atpdependent dna ligase 10. Set up the following reaction in a microcentrifuge tube on ice. Promega corporation 2800 woods hollow road madison, wi 537115399 usa. Minimum passing specification is dna repair, and recombination. 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 acids.

T4 dna ligase the enzyme efficiently joins blunt and. Identification and remediation of nucleotide bias in ssdna ligation is crucial because such bias can fail to. T4 dna ligase bluewhite cloning qualified protocol promega. Activities of enzymes commonly used in cloning applications. Each promega product is shipped with documentation. 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. 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. T4 dna ligase catalyzes the joining of two dna strands between the 5. It also joins dna fragments with either cohesive or blunt term.

As with t4 dna ligase, blunt end ligation is enhanced by the addition of peg 6000 to the reaction. This enzyme joins the break in the dna backbone structure by. 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. Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix.

Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. Incubate ligation reactions containing vector with blunt ends at 25c for 15 minutes. The roche t4 dna ligase manual im using says ligation should be kept for 16h. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector. 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. T4 dna ligase is used in molecular cloning experiments to ligate join the. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. Use only the t4 dna ligase supplied with this system to perform pgem t and pgemt easy vector ligations.