The ligafast rapid dna ligation system is designed for the efficient ligation of cohesiveended dna inserts into plasmid vectors in just 5 minutes bluntended inserts in as little as 15 minutes. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja. Cloning protocol dasen lab standard cloning with quick. The module is optimized for use with the nebnext end repair module and the nebnext datailing module and is part of the original standard dna library prep workflow, which is suitable for 1 5. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dna. Each promega product is shipped with documentation. 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. Ligafast rapid dna ligation system protocolpdf promega.
The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna. T4 dna ligase ligase dna ligation promega corporation. T4 dna ligase catalyzes the joining of two dna strands between the 5. 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.
The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. Xna ligation using t4 dna ligase in crowding conditions. Shop online for a wide selection of promega t4 dna ligase for ligation of dna ends. Promega ligafast rapid dna ligation system 2x rapid. Manual, second edition, cold spring harbor laboratory press, cold spring harbor. Straight lines indicate dna molecules, wavy lines indicate rna molecules and arrowheads.
Each microgram of cut plasmid is ligated with 4 units of t4 dna ligase. Ligation protocol with t4 dna ligase m0202 protocols. 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. The enzyme will not join singlestranded nucleic acids.
Comprehensive profiling of four base overhang ligation. Omerna duplexes, hna, lna and fana mixed sequences in the presence of 10% wv peg8000 and 3 m betaine. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. T4 rna ligase catalyzes the joining of singlestranded nucleic acids, including rna dna hybrids. Thermo scientific rapid dna ligation kit enables fast stickyend or bluntend dna ligation in only 5 minutes at room temperature. In this webinar, neb scientist and ligase expert greg lohman discusses mismatch ligation by dna ligases and the molecular diagnostics applications that depend on the use of highfidelity dna ligases like nebs hifi taq dna ligase to detect single base differences in dna. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. The kit contains t4 dna ligase and a speciallyformulated 5x rapid ligation buffer optimized for fast and efficient dna. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. The enzyme, purified from recombinant escherichia colica4 rnase i. T4 polynucleotide kinase catalyzes the transfer of the. T4 dna ligase is provided with 10x reaction buffer. Set up the following reaction in a microcentrifuge tube on ice.
T4 dna ligase is the industry standard for performance and quality. Learn more about how this product is being used in the. 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, bluewhite cloning qualified protocolpdf 112 kb english.
Page formattingtable formatting, incorrectmissing document pdf, other. Rapid ligation is based on the combination of t4 dna ligase. T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. Activities of enzymes commonly used in cloning applications. Improved amplification with a choice of buffers citations ning, b. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. 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.
Description t4 dna ligase catalyzes the joining of two strands of dna. The roche t4 dna ligase manual im using says ligation should be kept for 16h. T4 dna ligase bluewhite cloning qualified protocol promega. T4 dna ligase can catalyze the joining of rna to either a dna. The two dna ligases share many properties, but also have some distinct properties that. Dna ligase is a specific type of enzyme, a ligase, ec 6. Dna ligases are primarily responsible for joining the gaps that form in dna during replication.
Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Promega enzyme resource guide, cloning enzymes, br075b. A protocol for combining two strands of dna between the 5. The performance of this buffer depends on the integrity of the atp.
T4 dna ligase rapid the enzyme efficiently joins blunt. T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Toll free in the usa 8003569526 telephone 6082744330 internet. This chapter focuses on two of the best characterized dna ligases, the e. For this reason t4 dna ligase has become more widely used in dna manipulations table 1. For intermolecular ligations it is important that at least one of the dna molecules possesses a 5. 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. Promega products are warranted to meet or exceed the. These polymerases often add a single deoxyadenosine, in a template. Regulation of inter and intramolecular ligation with t4. T4 dna ligase is used in molecular cloning experiments to ligate join the. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5.
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