Ligation buffer optimized for fast and efficient dna ligation. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. L this protocol is for the dna insert ligation stickyend and bluntend into vector dna. T4 dna ligase recombinant form of the enzyme from t4 phage. Other suppliers may use cohesiveend ligation units. Materials in addition to t4 dna ligase and the insert and vector. T4 dna ligase, bluewhite cloning qualified protocolpdf 112 kb english. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Singlestranded nucleic acids are not substrates for this enzyme. Generating recombinant dna clones with anza t4 dna ligase. T4 dna ligase is used in molecular cloning experiments to ligate join the. Synthetized dna is cloned in blueheron puckan minus mcs vector.
One weiss unit of t4 ligase catalyzes the exchange of 1 nmol 32plabeled pyrophosphate into atp in 20 min at 37c 8. Centrifuge briefly to bring the contents to the bottom of the tube. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dna rna hybrids 1. L t4 dna ligase 5 u water, nucleasefree total volume 20. It also joins dna fragments with either cohesive or blunt termini. Dna insert ligation stickyend and bluntend into vector dna.
T4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed. Minimum passing specification is 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. Please visit anza to see our white paper on dephosphorylation using invitrogen anza alkaline. T4 dna ligase ligase dna ligation promega corporation. T4 dna ligase 5 ul, el0011, thermo fisher scientific 1. Assays formation of an enzymeadenylate intermediate this assay depends on the enzymes ability to covalently bind amp. Dna ligase, which is expressed in the early stage of lytic development, has been implicated in t4 dna synthesis and genetic recombination. The electrotransformation efficiency may be improved by. Nanodrop is a trademark of thermo fisher scientific inc. T4 dna ligase can be used to join dna fragments with staggered or blunt ends and to repair nicks in doublestranded dna having 3hydroxyl and 5phosphate.
T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3. Linear vector dna 20100 ng insert dna over vector thermo scientific 10x t4 dna ligase buffer 2. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna. T4 dna ligase can be used to join dna fragments with staggered or blunt ends and to repair nicks in doublestranded dna having 3hydroxyl and. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. T4 dna ligase will seal nicks for these dna substrates.695 561 1178 683 376 1224 660 473 665 613 311 1473 342 438 1013 1573 10 1018 1095 1368 876 770 913 265 99 124 1194 1043 1500 763 1242 631 682 689 418 198 974 155 523 1197 124 1434 1266 520 1474