Dna ligase reconnects dna strands when they are broken. Decades of expertise in enzymology, the large selection of enzyme. 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 this we utilized t4 dna ligase promega under the standard manufacturers conditions. Promega enzyme resource guide, cloning enzymes, br075b. For cohesive sticky ends, use 1 l of t4 dna ligase in a 20 l reaction for 10 minutes. A similar structure, that of t7 dna ligase, has been solved subramanya et al. Additional resources for t4 dna ligase technical bulletins and manuals 9pim180 t4 dna ligase promega product information the ligafast rapid dna ligation system cat. Through the fastdigest collection, you have access to one of the largest collections of restriction enzymes in the industry. If the dna concentrations are low such that you cannot get all 100ng of dna, buffer and ligase into a 10. T4 dna ligase is provided with 10x reaction buffer. Dna ligases catalyze the formation of a phosphodiester bond between dna single strands in the duplex form. Ligations using promega t4 dna ligase see product info sheet for more information. L of ligation master mix to each well of a new pcr plate.
T3 dna ligase was also found to have a higher efficiency in joining at matched bluntended. Overview protocols specifications resources t4 dna ligase catalyzes the joining of two cohesive or bluntended 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 ligase dna ligation promega corporation. Structural biochemistryt4 dna ligase wikibooks, open. 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. Unlike t4 rna ligase 1, t4 rna ligase 2 is much more active joining nicks on double. The t4 dna ligase is a single polypeptide with a molecular weight of 68,000 daltons. The performance of this buffer depends on the integrity of the atp.
I am looking for a protocol so that i can do this myself if anyone has had success. Vector dna 100 ng insert dna from equation ligase 10x buffer 1 l t4 dna ligase weiss units 0. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the 5. 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 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. Use of domain enzymes from wheat rna ligase for in vitro. T4 dna ligase is the industry standard for performance and quality. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use.
In contrast, atp is the cofactor for dna ligases from bacteriophages e. The temperature dependence of the t4 dna ligasecatalyzed joining of plasmid dna linearized by the action of haeiii, ecori and psti. In addition, it catalyzes the joining of duplex dna molecules at completely basepaired ends 2. Dna ligase is a specific type of enzyme, a ligase, ec 6. The impetus for our previous report silverman 2004 was the earlier finding by chiuman and li 2002 that 5. T4 dna ligase from promega,t4 dna ligase catalyzes the joining of two strands of dna between the 5. On the other hand, t4 dna ligase has the capacity to ligate bluntended dna without the aid of peg 6000, although the addition of 10% peg6000 can increase ligation efficiency greatly 4, 7. Incubate the reaction at room temperature for 3 hours, or at 4c overnight. Singlestranded nucleic acids are not substrates for this enzyme. The enzyme will not join singlestranded nucleic acids.
Our dna ligases and the dna ligase from the bacteriophage t7 shown at the top from pdb entry 1a0i use atp as the cofactor. 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. Herein we report the unexpectedly efficient ligation of rnasplinted dna by chlorella virus dna ligase pbcv1 dna ligase. T4 dna ligase buffer contains atp, so repeated freeze thaw cycles can degrade atp, thereby decreasing the efficiency of ligation. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. The exonuclease reaction was performed in t4 dna ligase buffer t4 buffer. This characteristic is quite similar to that of t7 dna ligase. 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. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. The enzyme efficiently joins blunt and cohesive ends and. The optimal insert to vector dna ratio, is usually between 2.
Set up the following reaction in a microcentrifuge tube on ice. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. On the basis of an analogous approach to dna 59adenylation using t4 dna ligase chiuman and li 2002 and our own procedure for rna 59adenylation with t4 rna ligase silverman 2004, here we. On ice, prepare the reaction mastermix with the following components. In these buffers t3 dna ligase exhibits an approximately 10fold reduction in activity. Structural biochemistryt4 dna ligase wikibooks, open books. Following incubation, the supercoiled dna is visualized on an ethidium bromidestained agarose gel. Characterization of bacteriophage t3 dna ligase the journal.
The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1. Both dna and rna ligases catalyze the formation of a phosphodiester bond between adja. Please remember to supplement the reaction with 1 mm atp final concentration. To test for endonuclease activity, 1g of type i supercoiled plasmid dna is incubated with 20 units of t4 dna ligase in 1x rapid ligation buffer for 16 hours at 37c. L of purified pcr product from the pcr cleanup plate, 2. Appdna can be synthesized in a related fashion by using 5. Ligation protocol with t4 dna ligase m0202 protocols. There must be no visible nicking or cutting of the dna. Toll free in the usa 8003569526 telephone 6082744330 internet usage information i. T4 rna ligases 1 and 2 are useful tools for rna analysis. 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. T4 dna ligase for t4 dna ligation, ta cloning, and other. Some engineering has been done to improve the in vitro activity of t4 dna ligase.
It is better to vortex or spin the t4 dna ligase enzyme before. T4 dna ligase catalyzes the joining of two strands of dna between the 5phosphate and the 3hydroxyl groups of adjacent nucleotides in either a. T4 dna ligase catalyzes the formation of a phosphodiester bond between 5 phosphate and 3 hydroxyl termini in duplex dna or rna. The domain symbols may be used in this paper to denote these enzymes. A simple method to obtain the 5 ends of mrna sequences by. A protocol for combining two strands of dna between the 5. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Our teams are in regular contact with suppliers and.
Incubate the reaction at room temperature for 3 hours. These activities of t4 dna ligase have been used to synthesize dna with defined sequences and to construct recombinant dna molecules in vitro. Promega corporation 2800 woods hollow roadmadison, wi 537115399 u. Full text get a printable copy pdf file of the complete article 1. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Ligation products were visualized on a 10% denaturing. T4 dna ligase catalyzes the joining of two cohesive or bluntended strands of dna between the. T4 dna ligase catalyzes the joining of two strands of dna between the 5. Pbcv1 dna ligase ligated ssdna splinted by rna with k cat. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or. 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.
The ligase 10x buffer supplied with this enzyme has a composition of 300mm trishcl ph 7. Their use upstream of rna analyses such as highthroughput rna sequencing and microarrays has recently increased their. 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. Fastdigest restriction enzymesthermo scientific us. L of ligase should be sufficient for larger ligation reactions.
T4 dna ligase 10x t4 dna ligase buffer 50% peg solution notes binding of t4 dna ligase to dna may result in a band shift in agarose gels. Unit definition one unit is defined as the amount of enzyme required to give 50% ligation of hindiii fragments of. For use with application joining doublestranded dna molecules with cohesive or blunt ends 5, 3 or bluntended dna inserts components. T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna.
Mutant t4 dna polymerase for easy cloning and mutagenesis. T4 rna ligase catalyzes the atpdependent ligation of singlestranded rna or dna onto the 5. The fulllength wheat rna ligase and its domain enzymes were prepared as in the previous study. Automated robotic liquid handling assembly of modular dna. Singlestranded dna library preparation from highly degraded. It uses a cofactor molecule shown in red for power and a special lysine amino acid shown in magenta to perform the reaction. Efficient dna ligation in dnarna hybrid helices by.
Purification of the t4 dna ligase by blue sepharose. T4 dna ligase bluewhite cloning qualified protocol promega. L reaction, scale the reaction size as necessary being sure to increase the amount of buffer proportionally. Higher concentrations of dna reaction components will result in a higher rate of reaction. Singlestranded dna ligation with t4 dna ligase and circligase.
It is better to vortex or spin the t4 dna ligase enzyme before pipetting to ensure that it is mixed well. For these purposes, the highly purified preparation of t4 dna ligase is necessary. I am looking for anyone who expresses their own enzymes rather than buys them from neb or promega. Ligafasttm rapid dna ligation system product information. To avoid this, incubate samples with 6x dna loading.
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