T4 dna ligase synthesizes dinucleoside polyphosphates core. One tube containing 2,500 u 5 u l is provided along with 10x reacti. It functions as a molecular glue to join nucleotides by forming phosphodiester bonds between nucleotides. 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. 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. 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. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master. In addition to t4 dna ligase and the insert and vector dnas, the following reagents and equipment are required for the protocols described below.
Ligation protocol with t4 dna ligase m0202 protocols. Ambion t4 rna ligase catalyzes the formation of a phosphodiester linkage between a 5phosphorylterminated nucleic acid donor and a 3hydroxylterminated nucleic acid acceptor. Invitrogen 15224017 view more versions of this product. Oh of the partner dna, even though the partner was 5. Rapid ligation protocol for plasmid cloning of dna fragments. Dna ligation is commonly used in molecular cloning projects to physically join a dna vector to a gene of interest. This products resides on a fisher scientific gsa or va contract. Dna ligase is a specific type of enzyme, a ligase, ec 6.
Expresslink t4 dna ligase formulation is optimized for faster reaction times and more convenient incubation temperature than our other t4 dna ligase formulations. L of purified pcr product from the pcr cleanup plate, 2. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. If the dna ligase is incubated with cxpiatp, the enzyme. The pl promoter from bacteriophage lambda under the control of a temperature. Control reaction for protocol t4 dna ligase activity. Increased cloning efficiency by temperaturecycle ligation.
One unit is equal to approximately 300 cohesiveend ligation units. The biphasic fermentations consisted of a primary growth phase at. One unit catalyzes the exchange of 1nmol 32 plabeled pyrophosphate into atp in 20 min. Longer incubation times increase the cloning efficiency, see the next page. Pdf functional characterization of the t4 dna ligase. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. 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. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. Centrifuge briefly to bring the contents to the bottom of the tube. To assess the activity of t4 dna ligase we recommend to perform ligation reaction with control dna, e. Yields of final ligation product for all reaction conditions using high concentration t4 dna ligase, the quick ligation kit and bluntta master mix. Singlestranded nucleic acids are not substrates for this enzyme. Ligafasttm rapid dna ligation system certificate of.
Difference between t4 and t7 dna ligase compare the. For high temperatures, we recommend taq dna ligase. Engineered dna ligases with improved activities in vitro protein. Pdf atpdependent dna ligases are essential enzymes in both dna. Singlestranded nucleic acids are not substrates for thi. The enzyme will not join singlestranded nucleic acids. Pdf t4 dna ligase is one of the workhorses of molecular biology and used in. Salt t4 dna ligase is insensitive to salt carried over from other reaction components vector or insert dna and allows ligation reactions to proceed in alternative reaction buffers with higher levels of salt e.
The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. 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. 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. 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.
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. All anza restriction enzymes come with an anza 10x clear buffer and an anza 10x red buffer to give you the flexibility you require. Digested plasmid dna was separated on 1% lowmelting agarose nusieve and purified as described by sambrook et al. T4 dna ligase ligase dna ligation promega corporation. For each digestion a ligation master mix was prepared containing 50 mm trishcl ph 7. Litmus 28i vector was cut with either ecorv blunt or hindiii cohesive, treated with calf intestinal phosphatase and gel purified. Rapid ligation is based on the combination of t4 dna ligase with a unique 2x rapid ligation buffer. T4 dna ligase is used in molecular cloning experiments to ligate join the ends of cloned dna fragments. Anza restriction enzymes thermo fisher scientific za. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Key difference t4 vs t7 dna ligase dna ligase is an important enzyme used in molecular biology techniques. The ends of the dna fragments can be blunt or cohesive and must contain monophosphate groups on the 5 ends. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini.
T4 dna ligase can ligate other substrates with reduced efficiency, including. Templateindependent ligation of singlestranded dna by t4. Download a pdf containing pricing for our full product list. The dna ligase from bacteriophage t4 is one of the most widely used enzymes in molecular biology. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes 1. Incubate the ligation reaction at room temperature for a minimum of 15 minutes. The phosphodiester bonds are formed between the 3 hydroxyl end of the sugar moiety and the 5 end phosphate group. Gently mix the reaction by pipetting up and down and microfuge briefly. 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. The quick ligation kit enables ligation of cohesive end or blunt end dna fragments in 5 minutes at room temperature. T4 dna ligase is one of the workhorses of molecular biology and used in various biotechnological applications.
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