{"id":1808,"date":"2021-06-05T16:45:36","date_gmt":"2021-06-05T16:45:36","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=1808"},"modified":"2021-06-05T17:14:14","modified_gmt":"2021-06-05T17:14:14","slug":"ta-and-topo-ta-cloning","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/services\/strategy-and-construct-design\/ta-and-topo-ta-cloning\/","title":{"rendered":"TA and TOPO\u00ae-TA cloning"},"content":{"rendered":"\n<div class=\"vf-grid | vf-grid__col-3\"><div class=\"vf-grid__col--span-2\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<p>The\u00a0<strong>TA cloning<\/strong>\u00a0method takes advantage of the terminal transferase activity of some DNA polymerases such as\u00a0<em>Taq<\/em>\u00a0polymerase. This enzyme adds a single, 3&#8242;-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3&#8242;-T overhangs. DNA polymerases with proofreading activity, such as\u00a0<em>Pfu<\/em>\u00a0polymerase, cannot be used because they provide blunt-ended PCR products.<\/p>\n\n\n\n<p>For example, the pTA plus vector (based on the pPCR-Script Amp from Stratagene) can be used for TA cloning. Digestion of this vector in two sequential reactions with&nbsp;<em>BamHI and XcmI<\/em> results in a linearized vector with 3&#8242;-T overhangs and a low background of non-recombinants.<\/p>\n\n\n\n<p>TA cloning is often combined with <strong>Topo\u00ae cloning<\/strong>, which takes advantage of the dual function of the enzyme DNA topo-isomerase I, which can act both as a restriction enzyme and a ligase. TOPO-TA vectors are usually already linearized and activated with topo-isomerase I, which allows direct ligation of PCR products with a 3\u2019-A overhang in 5 minutes. TOPO-TA vectors are available from <a href=\"https:\/\/www.thermofisher.com\/pt\/en\/home\/life-science\/cloning\/topo\/topo-ta-cloning.html\" target=\"_blank\" rel=\"noreferrer noopener\">Thermo Fischer Scientific<\/a>.<\/p>\n\n<\/div>\n<\/div>\n\n\n<div><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":971,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-1808","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1808","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/comments?post=1808"}],"version-history":[{"count":2,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1808\/revisions"}],"predecessor-version":[{"id":1846,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1808\/revisions\/1846"}],"up":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/971"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/media?parent=1808"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=1808"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}