Promega enzyme resource guide, cloning enzymes, br075b. 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. If the dna concentrations are low such that you cannot get all 100ng of dna, buffer and ligase into a 10. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. The ligase preparation was further subjected to the sephadex g100 chromatography according to raae et al. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. Promega vs neb t4 dna ligase as well as ligase buffer. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. Singlestranded nucleic acids are not substrates for thi. Templateindependent ligation of singlestranded dna by t4. L of ligation master mix to each well of a new pcr plate. Purification of the t4 dna ligase by blue sepharose.
T4 dna ligase bluewhite cloning qualified t4 dna ligase. Cloning protocol dasen lab standard cloning with quick ligase. T4 dna ligase in combination with the 2x rapid ligation buffer greatly stimulates the rate and efficiency of bluntend ligation, therefore long incubations 10 minutes are not recommended and can greatly reduce the transformation efficiency of ligation products. 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. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5.
Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. The enzyme most commonly used is derived from bacteriophage t4. Ligation of dna material to be supplied by the user nucleasefree water cat. T4 dna ligase catalyzes the joining of two strands of dna between the 5 phosphate and the 3hydroxyl groups of adjacent nucleotides in either a. 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. Ligafasttm rapid dna ligation system product information sheet.
T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna fragments with 3oh and 5phosphate ends, in the presence of atp. Cloning protocol dasen lab standard cloning with quick ligase neb preparation of dna. 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 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5.
In this study, we report results on the expression and purification of recombinant t4 dna ligase. Promega ligafast rapid dna ligation system 2x rapid. T4 dna ligase is provided with 10x reaction buffer. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. 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. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful.
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. 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 nucleic. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. Promega products are warranted to meet or exceed the. The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. Efficient synthesis of stably adenylated dna and rna. 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. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and. 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, 1 weiss ul 2x5 00 weiss u 10x t4 dna ligase buffer 1. Dna ligase is a specific type of enzyme, a ligase, ec 6. Test of bsmbi and t7 dna ligase procedure using neb buffer 3. L of purified pcr product from the pcr cleanup plate, 2. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in a duplex molecule but. Each promega product is shipped with documentation stating specifications and other technical information. T4 dna ligase catalyzes the joining of two strands of dna between the 5. The extent of ligation can be determined by estimation of the relative intensity of fluorescence in the gel bands of substrate and product. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. 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. Promega ligafast rapid dna ligation system 2x rapid ligation. Set up the following reaction in a microcentrifuge tube on ice.
T4 rna ligase is provided with 10x reaction buffer. 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. Vector dna 100ng insert dna ligase 10x buffer t4 dna ligase nucleasefree water protocol. For blunt ends or single base overhangs, incubate at 16c overnight or room temperature for 2 hours alternatively, high concentration t4 dna ligase can be used in a 10 minute ligation. Ligation protocol with t4 dna ligase m0202 protocols.
The flexi vector system is a directional cloning method for proteincoding sequences. Plasmid pet16bt4dnl contains the gp30 gene which encodes for t4 dna ligase. T4 dna ligase, bluewhite cloning qualified protocol pdf 112 kb english. Description t4 dna ligase catalyzes the joining of two strands of dna between the 5. 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. In order to maximize transformation efficiency of the correct insertvector. The enzyme will not join singlestranded nucleic acids. T4 dna ligase ligase dna ligation promega corporation. Promega ligafast rapid dna ligation system designed to eliminate the need for further purification prior to transformation of ligated dna. L of ligase should be sufficient for larger ligation reactions. Ligafasttm rapid dna ligation system product information.
Efficient synthesis of stably adenylated dna and rna adapters for microrna capture using t4 rna ligase 1. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. The performance of this buffer depends on the integrity of the atp. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and.
Toll free in the usa 8003569526 telephone 6082744330 internet. T4 dna ligase is used in molecular cloning experiments to ligate join the. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids. T4 dna ligase bluewhite cloning qualified protocol promega.
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. Cloning protocol dasen lab standard cloning with quick. A similar structure, that of t7 dna ligase, has been solved subramanya et al. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english.
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