Insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. 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. 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. Recombinant clones can be efficiently selected using bluewhite screening on indicator plates. Ive heard that both reagents can be substituted with promegas t4 dna ligase and buffer attached file to yield highquality results. 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. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja cent nucleotides. 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 ligase dna ligation promega corporation.
The enzyme will not join singlestranded nucleic acids. The insert dna can easily be removed for subcloning using an ecori single digest. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. Singlestranded dna ligation with t4 dna ligase and circligase. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. 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. 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. 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 pt arge t mammalian expression vector system includes jm109 high efficiency competent cells for transformation of ligation reactions. Structural biochemistryt4 dna ligase wikibooks, open books.
T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. For new targets, testing dilutions of donor dna will ensure optimal saturation of donor molecules for the best possible assay. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. 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. In order to obtain the maximum amount of activity from the ligase, a ph of 7. 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 performance of this buffer depends on the integrity of the atp.
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. Use only the t4 dna ligase provided with the system, as this has been tested. Dna ligase reconnects dna strands when they are broken. 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. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. T4 dna ligase is the industry standard for performance and quality. 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.
Learn more about how this product is being used in the product citation tool. 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. Instructions for use of products m1801, m1804, m1794. Ligation protocol with t4 dna ligase m0202 protocols. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. T4 dna ligase is provided with 10x reaction buffer.
T4 dna ligase bluewhite cloning qualified protocol promega. The roche t4 dna ligase manual im using says ligation should be kept for 16h. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. 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. 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. Toll free in the usa 8003569526 telephone 6082744330 internet. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks inl6030 duplex dna, rna or dna rna hybrids 1. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. For details on nebs quality controls for dna ligases, visit our ligase quality page. 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. Fastdigest restriction enzymesthermo scientific thermo. 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 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. Dna ligase is a specific type of enzyme, a ligase, ec 6. 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 phosphodiester bonds in the presence of atp between doublestranded dnas with 3. T4 dna ligase catalyzes the templatedependent ligation of dna. Cloning protocol dasen lab standard cloning with quick ligase neb preparation of dna.
The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. Canonical nucleosides can be utilized by t4 dna ligase as. For this reason t4 dna ligase has become more widely used in dna manipulations table 1. The two dna ligases share many properties, but also have some distinct properties that make them uniquely useful depending on application. Singlestranded dna library preparation from highly degraded. Pathway complexity in fueldriven dna nanostructures with. Set up the following reaction in a microcentrifuge tube on ice. 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. For intermolecular ligations it is important that at least one of the dna molecules possesses a 5. 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 polymerase catalyzes the synthesis of dna in the 5.
Bacteriophage t4 dna ligase is the ligase most commonly used in the construction of recom. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. Singlestranded nucleic acids are not substrates for this enzyme. Cloning protocol dasen lab standard cloning with quick. Fiveminute incubations at 25c are used for kpni and ecor i, and 15 minute incubations at. T4 polynucleotide kinase catalyzes the transfer of the. Po4 at the ends of two polynucleotide chains frequently at a nick in one strand of a double helix. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. Please remember to supplement the reaction with 1 mm atp final concentration. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english.
Ligation products were visualized on a 10% denaturing. The fastdigest green buffer and thermo scientific fastdigest buffer are proprietary digestion buffers which support 100% activity of all fastdigest restriction enzymes. T4 dna ligase rapid the enzyme efficiently joins blunt. The enzyme may be used to phosphorylate rna, dna and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Ligafasttm rapid dna ligation system product information. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5. A ligation control dna is included as a system check. Dna ligase is an enzyme that forms a covalent phosphodiester bond between a 3.
The t4 dna polymerase produced in this fashion was purified by an innovative threestep procedure and was fully active. 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. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Promega manufactures products for a number of intended uses. This chapter focuses on two of the best characterized dna ligases, the e. Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. Promega enzyme resource guide, cloning enzymes, br075b. 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 catalyzes the formation of a phosphodiester bond between the terminal 5. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. L aliquots of the reaction solution were collected and quenched by 4. It also joins dna fragments with either cohesive or blunt term. 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 can be used to join dna fragments with staggered or blunt ends. 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. 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. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5.