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. Neb offers many different kinds of dna and rna ligases for your needs. A previously reported method used double stranded t4 dna ligase to perform enzymatic adenylation 9. The enzyme may be used to phosphorylate rna, dna and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. This chapter focuses on two of the best characterized dna ligases, the e. In this study, we report results on the expression and purification of recombinant t4 dna ligase.
The addition of polyethylene glycol peg to ligation reactions can promote ligation of bluntended fragments by macromolecular crowding 2. This chart will help you understand the differences between them. A pool of 60 nt acceptor oligonucleotides 60n were ligated to 10 pmol of a 3. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. For details on nebs quality controls for dna ligases, visit our ligase quality page. 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. T4 dna ligase is routinely used to ligate two rna strands using a dna splint. 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.
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. 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. Checking your t4 dna ligase activity thermo fisher. T4 dna ligase bluewhite cloning qualified protocol promega. T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. Shop online for a wide selection of promega t4 dna ligase for ligation of dna ends. A ligation control dna is included as a system check.
Set up the following reaction in a microcentrifuge tube on ice. Dna ligase from t4infected escherichia coli and promega t4 dna ligase buffer. T4 dna ligase is commonly employed in these methodsdue to its high e. Promega for quality control of lambda vectors with sticky ends. Efficiently ligates dna inserts into plasmid vectors in just 515 minutes at room temperature. The enzyme, purified from recombinant escherichia coli ca4 rnase ideficient, has an apparent molecular weight of 43. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase. Promega products are warranted to meet or exceed the. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Toll free in the usa 8003569526 telephone 6082744330 internet.
T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications. 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 5. These ligation conditions have been developed using promega bluewhite cloningqualified t4 dna ligase. Fiveminute incubations at 25c are used for kpni and ecor i, and 15 minute incubations at. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. Each promega product is shipped with documentation stating specifications and other technical information. In addition, dna ligase selection chart helps you select the optimal dna ligase for various applications. Dna ligase i is more effective at bluntend joining than mam malian dna ligases i1 and 111 but is less efficient in this regard than bac teriophage t4 dna ligase. This dna is highly pure and should be easily ligated using the protocol below.
Vector dna 100ng insert dna ligase 10x buffer t4 dna ligase nucleasefree water protocol. 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. 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. A protocol for combining two strands of dna between the 5. Singlestranded dna library preparation from highly degraded. T4 dna ligase is provided with 10x reaction buffer. Ligation protocol with t4 dna ligase m0202 protocols. Ligafasttm rapid dna ligation system product information sheet. The kit also contains polyethylene glycol peg 8000, which enhances bluntended ligations by macromolecular crowding. Ligation products were visualized on a 10% denaturing. T4 dna ligase is used in molecular cloning experiments to ligate join the.
If the dna ligase is incubated with cxpiatp, the enzyme. 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. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. Promega enzyme resource guide 6 two when doing bluntend ligation or single restriction enzyme site ligation, it is advisable to. Dilutions of enzyme batch were made in 1x t4 dna ligase. The ideal temperature for the t4 dna ligase reaction is 16 qc, however it works over a range of temperatures, so 4 qc or room temperature can also be used. 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 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. The ligation reaction mixture was incubated at room temperature for 15 min, purified using qiaquick pcr purification kit, and eluted with 80.
T4 polynucleotide kinase catalyzes the transfer of the. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. Promega enzyme resource guide 8 two ligases introduction dna ligases are primarily responsible for joining the gaps that form in dna during replication i. 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. Promega ligafast rapid dna ligation system 2x rapid ligation buffer life sciences. 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 rna ligase catalyzes the joining of singlestranded nucleic acids, including rna dna hybrids. Templateindependent ligation of singlestranded dna by t4. 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. Dna ligase is a specific type of enzyme, a ligase, ec 6. Comprehensive profiling of four base overhang ligation. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. Ligafast rapid dna ligation system protocol promega. The enzyme will not join singlestranded nucleic acids. Use only the t4 dna ligase supplied with this system to perform pgem t and pgemt easy vector ligations. 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. A method to convert mrna into a grna library for crispr. Instructions for use of products m1801, m1804, m1794.
The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. T4 dna ligase bluewhite cloning qualified t4 dna ligase. The two dna ligases share many properties, but also have some distinct properties that make them uniquely useful depending on application. Learn more about our custom options for this product at. Unit activity was measured using a 2fold serial dilution method.
T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5. Ligafasttm rapid dna ligation system product information. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5. The pgemt vector systems are convenient systems to clone pcr products generated by certain thermostable polymerases. 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. If the dna is not ligated you may want to check the activity of your ligase by using lambda dna digested with hindiii cat. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3. T4 dna ligase ligase dna ligation promega corporation. Here we report that this ligase, unlike escherichia coli dna ligase, taq dna ligase and ampligase, is able to join the ends of singlestranded dna in the absence of any duplex dna structure at the ligation site. Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector. Other commercial preparations of t4 dna ligase may contain exonuclease activities that may remove.
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. Promega ligafast rapid dna ligation system 2x rapid. T4 dna ligase is a ligation enzyme that can be used to join dna fragments by catalyzing the formation of phosphodiester bonds between juxtaposed 5 phosphate and 3 hydroxyl termini in doublestranded dna using atp as a coenzyme. Promega enzyme resource guide, cloning enzymes, br075b.
However, this enzyme is wellknown to join mismatches, gaps, and other imperfect structures with varying levels of e. We postulated that by switching to single stranded t4. Use only promega t4 dna ligase supplied with this system to perform. 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. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. Identification of the substancepreparation and of the companyundertaking product name t4 dna ligase product no m0202 recommended use of the chemical and restrictions on use. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 rna ligase catalyzes the joining of singlestranded nucleic acids, including rnadna hybrids.
T4 dna ligase, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. This section describes the general properties of the two enzymes. Safety data sheet document type aghs osha ghs revision date 21jul2016 version 3 1. Efficient synthesis of stably adenylated dna and rna. Promega ligafast rapid dna ligation system 2x rapid ligation. It also joins dna fragments with either cohesive or blunt term.
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