The ends of the dna fragments can be blunt or cohesive and must contain monophosphate groups on the 5 ends. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. Pdf functional characterization of the t4 dna ligase. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications. One unit is equal to approximately 300 cohesiveend ligation units. P, the fragment remained capable of ligating with the 3.
The phosphodiester bonds are formed between the 3 hydroxyl end of the sugar moiety and the 5 end phosphate group. The enzyme will not join singlestranded nucleic acids. The pl promoter from bacteriophage lambda under the control of a temperature. There is considerable latitude in the temperature and time needed for successful ligations.
Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. To assess the activity of t4 dna ligase we recommend to perform ligation reaction with control dna, e. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. T4 dna ligase catalyzes the formation of phosphodiester bonds between doublestranded dna strands with 3 hydroxyl and 5 phosphate termini in the presence of atp. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. Centrifuge briefly to bring the contents to the bottom of the tube. Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. The primary structure of phage t4 dna ligase has been determined by dna sequencing of a cloned restriction fragment containing its gene, and partial amino acid sequence analysis of the protein. T4 dna ligase synthesizes dinucleoside polyphosphates core. For high temperatures, we recommend taq dna ligase. Control reaction for t4 dna ligase activity unsuccessful ligation may be a result of inactivated ligase or inhibition of ligation reaction by impurities in sample dna.
Bamhi site of the ptrchisa expression vector invitrogen. Control reaction for protocol t4 dna ligase activity. The infection with a t4 mutant, t4awh39x which is defective in the dna ligase gene, enables preparation of rna ligase free of dna ligase 1. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Difference between t4 and t7 dna ligase compare the. Incubate the ligation reaction at room temperature for a minimum of 15 minutes. Gently mix the reaction by pipetting up and down and microfuge briefly. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min. In addition to t4 dna ligase and the insert and vector dnas, the following reagents and equipment are required for the protocols described below. It functions as a molecular glue to join nucleotides by forming phosphodiester bonds between nucleotides. Ambion t4 rna ligase catalyzes the formation of a phosphodiester linkage between a 5phosphorylterminated nucleic acid donor and a 3hydroxylterminated nucleic acid acceptor. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Use of t4 phage mutants dorega enables one to overproduce the ligase to as much as sevenfold over the amount of wild type or 0.
Pdf t4 dna ligase is one of the workhorses of molecular biology and used in. The red buffer includes a density reagent containing red and yellow tracking dyes that migrate with 800 bp dna fragments and faster than the 10 bp dna fragments, respectively, in a. Singlestranded nucleic acids are not substrates for thi. Dna ligase catalyzes the formation of the phosphodiester bond between a 5. 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. Key difference t4 vs t7 dna ligase dna ligase is an important enzyme used in molecular biology techniques. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Ligafasttm rapid dna ligation system certificate of. Invitrogen 15224017 view more versions of this product. One tube containing 2,500 u 5 u l is provided along with 10x reacti. Pdf atpdependent dna ligases are essential enzymes in both dna. All anza restriction enzymes come with an anza 10x clear buffer and an anza 10x red buffer to give you the flexibility you require. T4 dna ligase ligase dna ligation promega corporation.
Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master mix. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. 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. 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. Oh of the partner dna, even though the partner was 5. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. T4 dna ligase is used in molecular cloning experiments to ligate join the ends of cloned dna fragments. Dna ligase is a specific type of enzyme, a ligase, ec 6. If the dna ligase is incubated with cxpiatp, the enzyme. Engineered dna ligases with improved activities in vitro protein. Templateindependent ligation of singlestranded dna by t4. T4 dna ligase can ligate other substrates with reduced efficiency, including. The biphasic fermentations consisted of a primary growth phase at.
For each digestion a ligation master mix was prepared containing 50 mm trishcl ph 7. Anza restriction enzymes thermo fisher scientific za. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master. The dna ligase from bacteriophage t4 is one of the most widely used enzymes in molecular biology.
T4 dna ligase is provided with 10x reaction buffer. Dna ligation is commonly used in molecular cloning projects to physically join a dna vector to a gene of interest. Rapid ligation protocol for plasmid cloning of dna fragments. Download a pdf containing pricing for our full product list. Longer incubation times increase the cloning efficiency, see the next page. Set up the following reaction in a microcentrifuge tube on ice. 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. Ligation protocol with t4 dna ligase m0202 protocols.