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. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. T4 dna ligase bluewhite cloning qualified protocol promega. Learn more about how this product is being used in the product citation tool. 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 insert dna can easily be removed for subcloning using an ecori single digest. Unlike t4 rna ligase 1, t4 rna ligase 2 is much more active joining nicks on double stranded rna than on joining the ends of single stranded rna. The t4 dna polymerase produced in this fashion was purified by an innovative threestep procedure and was fully active.
Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5. T4 dna ligase catalyzes the joining of two strands of dna between the 5. T4 dna ligase is used in molecular cloning experiments to ligate join the ends of cloned dna fragments, ligase is commonly to insert dna into a cloning. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. Reaction conditions 2x rapid ligation buffer t4 dna ligase 10x buffer 1 hour at 24c 259 29 16 hours at 4c 580 164 cloning efficiency was calculated as the number of white coloniesng vector dna from a ligation containing 8ng control insert dna 542bp and 50ng pgemt easy 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. T3 dna ligase is also active in buffers without peg 6000, such as our t4 dna ligase buffer and nebuffers 14, for applications in which peg 6000 is detrimental. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. L aliquots of the reaction solution were collected and quenched by 4. This chapter focuses on two of the best characterized dna ligases, the e.
It also joins dna fragments with either cohesive or blunt term. This system allows for double and multiple digestions with any combination of enzymes. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. The roche t4 dna ligase manual im using says ligation should be kept for 16h. Fastdigest restriction enzymesthermo scientific thermo. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. Cloning protocol dasen lab standard cloning with quick ligase neb preparation of dna. 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. Ligafasttm rapid dna ligation system product information. The enzyme will not join singlestranded nucleic acids. Specifically formulated to rapidly ligate cohesiveend 24 bp substrates and improve transformation, instant stickyend ligase master mix is a readytouse 2x solution of t4 dna ligase and a proprietary ligation enhancer in an optimized reaction buffer.
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. It is based on two rarecutting restriction enzymes, sgfi and pmei, and provides a rapid, efficient and highfidelity way to transfer proteincoding regions without the need to resequence. Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. Singlestranded dna library preparation from highly degraded. The fastdigest green buffer and thermo scientific fastdigest buffer are proprietary digestion buffers which support 100% activity of all fastdigest restriction enzymes. For details on nebs quality controls for dna ligases, visit our ligase quality page. At a 1x concentration this reaction buffer assures optimal activity of the enzyme.
Full text get a printable copy pdf file of the complete article 1. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. Both companies tech support provided inconclusive answers. Fiveminute incubations at 25c are used for kpni and ecor i, and 15 minute incubations at. Canonical nucleosides can be utilized by t4 dna ligase as. A ligation control dna is included as a system check. Structural biochemistryt4 dna ligase wikibooks, open books. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Toll free in the usa 8003569526 telephone 6082744330 internet. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature.
Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. Pathway complexity in fueldriven dna nanostructures with. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Use only the t4 dna ligase provided with the system, as this has been tested. T4 dna ligase ligase dna ligation promega corporation.
Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. T4 polynucleotide kinase catalyzes the transfer of the. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja cent nucleotides. In order to obtain the maximum amount of activity from the ligase, a ph of 7. Dna ligase is a specific type of enzyme, a ligase, ec 6. Cloning protocol dasen lab standard cloning with quick. 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. T4 dna ligase is provided with 10x reaction buffer. T4 dna ligase rapid the enzyme efficiently joins blunt. Singlestranded nucleic acids are not substrates for this enzyme. The two dna ligases share many properties, but also have some distinct properties that make them uniquely useful depending on application. The performance of this buffer depends on the integrity of the atp. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
Promega manufactures products for a number of intended uses. Genetic engineering is a modern biotechnology technique which is used to modify the genetic makeup of an organism by adding new traits in to it and there by produce new variety of organisms. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5.
A pool of 60 nt acceptor oligonucleotides 60n were ligated to 10 pmol of a 3. Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3. 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. For intermolecular ligations it is important that at least one of the dna molecules possesses a 5. The pt arge t mammalian expression vector system includes jm109 high efficiency competent cells for transformation of ligation reactions. Please remember to supplement the reaction with 1 mm atp final concentration. 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. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Ligation products were visualized on a 10% denaturing. T4 dna ligase catalyzes the templatedependent ligation of dna. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english.
Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. T4 dna polymerase catalyzes the synthesis of dna in the 5. Ligation protocol with t4 dna ligase m0202 protocols. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. T4 dna ligase is the industry standard for performance and quality. The enzyme may be used to phosphorylate rna, dna and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning. Instructions for use of products m1801, m1804, m1794. 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. Bacteriophage t4 dna ligase is the ligase most commonly used in the construction of recom. Genetic engineering is a modern biotechnology technique which is used to modify the genetic makeup of an organism by adding new traits in to it and there by produce new variety of organ.
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. 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. 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. Promega enzyme resource guide, cloning enzymes, br075b. Ive heard that both reagents can be substituted with promegas t4 dna ligase and buffer attached file to yield highquality results. Recombinant clones can be efficiently selected using bluewhite screening on indicator plates. T4 rna ligase 1 catalyzes the ligation of a 5 phosphorylterminated nucleic acid donor to a 3 hydroxylterminated nucleic acid acceptor through the formation of a 3. 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. 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. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dna rna hybrids 1.
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