Thermolabile Alkaline Phosphatase (TAP) catalyzes the dephosphorylation of 5′ and 3′ ends of DNA and RNA. Also, TAP hydrolyzes ribo- and deoxyribonucleoside triphosphates.
Applications:<\/b>
-removing 5` and 3` phosphoryl groups from nucleic acids;
-preparing templates for 5` end labeling;
-preventing DNA fragments from self-ligating.<\/p>\n
Source:<\/strong> Isolated from E.coli strain that carries the cloned Alkaline Phosphatase gene from Alteromonas undina P2<\/p>\n Unit definition:<\/strong> Thermolabile Alkaline Phosphatase (TAP) catalyzes the dephosphorylation of 5′ and 3′ ends of DNA and RNA. Also, TAP hydrolyzes ribo- and deoxyribonucleoside triphosphates. Reaction buffer:<\/strong> SE-buffer W<\/p>\n Storage conditions:<\/strong> 20 mM Tris-HCl (pH 7.6), 0.1 mM ZnCl2<\/sub>, 50% glycerol. Store at -20°C<\/p>\n Quality control:<\/strong> The enzyme is purified free of contaminating endonucleases andexonucleases, ribonuclease activities.<\/p>\n Notes:<\/strong> Functional assay:<\/b> As a result of treatment 1μg of pUC19\/Hind III DNA containing 1X SE-buffer W and 1μl of the enzyme in a total reaction volume of 20 μl in 30 min at 16°C , with following ligation with T4 DNA Ligase and transformation of E.coli cells, >10-times decreasing of clones amount was achieved compared to use of untreated pUC19\/Hind III DNA. Removes 5\u00b4- and 3\u00b4- phosphates from DNA, RNA and dNTPs. May be used instead of Antarctic Phosphatase<\/p>\n","protected":false},"featured_media":58,"template":"","meta":[],"product_brand":[],"product_cat":[498],"product_tag":[],"class_list":{"0":"post-2704","1":"product","2":"type-product","3":"status-publish","4":"has-post-thumbnail","6":"product_cat-enzymes","7":"pa_285b89be-c12827be1b","8":"pa_4ebaafd1-pa_4ebaafd_d66329-403","9":"pa_50b54da1-c74d97b01e","10":"pa_concentration-13be3a710f","11":"pa_fasovka-65253e40ca","12":"pa_fasovka-658e880e51","13":"pa_package-d20caec3b4","14":"pa_package-5dbc98dcc9","16":"first","17":"instock","18":"taxable","19":"shipping-taxable","20":"purchasable","21":"product-type-variable","22":"berocket_lgv_grid","23":"berocket_lgv_list_grid"},"_links":{"self":[{"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/product\/2704","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/product"}],"about":[{"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/types\/product"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/media\/58"}],"wp:attachment":[{"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/media?parent=2704"}],"wp:term":[{"taxonomy":"product_brand","embeddable":true,"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/product_brand?post=2704"},{"taxonomy":"product_cat","embeddable":true,"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/product_cat?post=2704"},{"taxonomy":"product_tag","embeddable":true,"href":"https:\/\/sibenzyme.com\/en\/wp-json\/wp\/v2\/product_tag?post=2704"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
Applications:<\/b>
-removing 5` and 3` phosphoryl groups from nucleic acids;
-preparing templates for 5` end labeling;
-preventing DNA fragments from self-ligating.<\/p>\n
Protocol for dephosphorilation<\/b> of 5′-ends of DNA digested with Restrction Endonuclease (RE) using TAP:
20 μl of the reaction volume:
10X SE-buffer for RE or Buffer W -2 μl
DNA – 0,5-1 μg
Nuclease-free water -to 20 μl
Add 1 μl of RE, mix by pipetting.
Incubate for 0,5-1 hour at optimal temperature.
Chill the reaction mixture on ice for 1-2 min!!!<\/b>Add 1 μl (5 units) of TAP (E365\/E366), mix by pipetting.Incubate for 0,5-1 hour at 16°C (Incubation at 25°C results in 75-100% activity).Heat the reaction mixture for 20 min at 65°C.If necessary, make precipitation of DNA with ethanol or purify it by gel-filtration or spin-column.
\n<\/p>","protected":false},"excerpt":{"rendered":"