{"id":9783,"date":"2022-02-28T11:25:53","date_gmt":"2022-02-28T11:25:53","guid":{"rendered":"https:\/\/cotslab.com\/uv-vis-spektrofotometrie-snadna-a-rychla-kvantifikace-nukleovych-kyselin\/"},"modified":"2022-02-28T11:25:58","modified_gmt":"2022-02-28T11:25:58","slug":"uv-vis-spektrofotometrie-snadna-a-rychla-kvantifikace-nukleovych-kyselin","status":"publish","type":"post","link":"https:\/\/cotslab.com\/cs\/uv-vis-spektrofotometrie-snadna-a-rychla-kvantifikace-nukleovych-kyselin\/","title":{"rendered":"UV-Vis spektrofotometrie \u2013 snadn\u00e1 a rychl\u00e1 kvantifikace nukleov\u00fdch kyselin"},"content":{"rendered":"\n<p>M\u011b\u0159en\u00ed absorbance roztoku nukleov\u00e9 kyseliny pouze p\u0159i \u010dty\u0159ech vlnov\u00fdch d\u00e9lk\u00e1ch je dostate\u010dn\u00e9 pro stanoven\u00ed jej\u00ed koncentrace a pro z\u00edsk\u00e1n\u00ed n\u00e1hledu na jej\u00ed \u010distotu.<\/p>\n\n<p><em>Zdroj obr\u00e1zk\u016f: DRogatnev\/shutterstock.com<\/em><\/p>\n\n<p>Nukleov\u00e9 kyseliny jsou izolov\u00e1ny ze vzorku materi\u00e1lu, jako jsou bu\u0148ky, a n\u00e1sledn\u011b pou\u017eity v dal\u0161\u00edch laboratorn\u00edch experimentech. Pro tento \u00fa\u010del je vhodn\u00e9 stanovit koncentraci purifikovan\u00e9 DNA nebo RNA a tak\u00e9 ov\u011b\u0159it jejich \u010distotu. T\u00edmto zp\u016fsobem p\u0159esn\u011b definovan\u00e1 mno\u017estv\u00ed nukleov\u00e9 kyseliny vstoup\u00ed do n\u00e1sledn\u00e9 aplikace a jak\u00e9koli kontaminace, kter\u00e9 mohou ovlivnit citlivou reakci nebo test, jsou detekov\u00e1ny v re\u00e1ln\u00e9m \u010dase.<\/p>\n\n<p>UV-Vis spektrofotometrie je snadn\u00e1, rychl\u00e1 a \u010dasem prov\u011b\u0159en\u00e1 metoda k dosa\u017een\u00ed t\u011bchto c\u00edl\u016f. V rozsahu 260 nm vykazuj\u00ed nukleov\u00e9 kyseliny charakteristick\u00fd p\u00edk absorbance (obr\u00e1zek 1). Tato hodnota absorbance se proto pou\u017e\u00edv\u00e1 k v\u00fdpo\u010dtu koncentrac\u00ed nukleov\u00e9 kyseliny. Podle Lambert-Beerova z\u00e1kona jsou pro v\u00fdpo\u010det koncentrace vzorku nutn\u00e9 dva parametry: d\u00e9lka optick\u00e9 dr\u00e1hy (= d\u00e9lka sv\u011bteln\u00e9 dr\u00e1hy (L)) a mol\u00e1rn\u00ed extink\u010dn\u00ed koeficient (konstanta specifick\u00e1 pro materi\u00e1l a vlnovou d\u00e9lku) vzorku. vzorek k m\u011b\u0159en\u00ed. Specifick\u00fd faktor (F) lze vypo\u010d\u00edtat p\u0159i pou\u017eit\u00ed standardn\u00ed kyvety s dr\u00e1hou sv\u011btla 1 cm. Tento faktor se pak vyn\u00e1sob\u00ed nam\u011b\u0159enou hodnotou absorbance (A), aby se dosp\u011blo ke koncentraci (C) roztoku vzorku. Mol\u00e1rn\u00ed extink\u010dn\u00ed koeficienty a specifick\u00e9 faktory jsou typicky dostupn\u00e9 v literatu\u0159e. Faktor pro dsDNA je nap\u0159\u00edklad 50 ug\/ml (RNA: 40 ug\/ml) a je podle definice ekvivalentn\u00ed jedn\u00e9 jednotce absorbance. tyto \u00fadaje.<\/p>\n\n<figure class=\"wp-block-table\"><table><tbody><tr><td><strong>Lambert-Beer\u016fv z\u00e1kon:<\/strong><br\/><br\/> <strong>C = A\/(L x \u0190)<\/strong><br\/> <strong>F = 1\/\u0190<\/strong> (pro dr\u00e1hu sv\u011btla 1 cm)<br\/><br\/>  <strong> &#8211; C = A x F<\/strong><\/td><td> C = koncentrace<br\/> A = Absorbance<br\/> L = d\u00e9lka optick\u00e9 (sv\u011bteln\u00e9) dr\u00e1hy<br\/> \u0190 = Mol\u00e1rn\u00ed extink\u010dn\u00ed koeficient<br\/> (specifick\u00e9 pro vzorek a vlnovou d\u00e9lku)<br\/> F = faktor<\/td><\/tr><\/tbody><\/table><\/figure>\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" width=\"768\" height=\"456\" src=\"https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_One_Rahmen_b55fd8fbd6.jpg\" alt=\"\" class=\"wp-image-9788\" srcset=\"https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_One_Rahmen_b55fd8fbd6.jpg 768w, https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_One_Rahmen_b55fd8fbd6-674x400.jpg 674w, https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_One_Rahmen_b55fd8fbd6-600x356.jpg 600w\" sizes=\"(max-width: 768px) 100vw, 768px\" \/><figcaption>Obr\u00e1zek 1: Absorban\u010dn\u00ed spektrum nukleov\u00fdch kyselin s relevantn\u00edmi vlnov\u00fdmi d\u00e9lkami a p\u0159\u00edklad popisuj\u00edc\u00ed v\u00fdpo\u010det koncentrace vzorku dsDNA.<\/figcaption><\/figure><\/div>\n\n<p>Aby bylo mo\u017en\u00e9 u\u010dinit prohl\u00e1\u0161en\u00ed o \u010distot\u011b vzorku nukleov\u00e9 kyseliny, je t\u0159eba ur\u010dit absorbanci p\u0159i vlnov\u00fdch d\u00e9lk\u00e1ch jin\u00fdch ne\u017e 260 nm (obr\u00e1zek 1). Kvocienty odvozen\u00e9 z hodnot absorbance nam\u011b\u0159en\u00fdch p\u0159i <sub>260<\/sub> nm, <sub>280<\/sub> nm a <sub>230<\/sub> nm (A260\/A280 a <sub>A260<\/sub> \/A230) tvo\u0159\u00ed pom\u011bry \u010distoty, kter\u00e9 pom\u00e1haj\u00ed identifikovat mo\u017en\u00e9 kontaminace. Ne\u010distoty, jako jsou proteiny a stopy reagenci\u00ed, kter\u00e9 byly pou\u017eity b\u011bhem procesu \u010di\u0161t\u011bn\u00ed, poskytuj\u00ed jin\u00e9 absorban\u010dn\u00ed spektrum ne\u017e spektrum nukleov\u00fdch kyselin a ovliv\u0148uj\u00ed tak pom\u011bry \u010distoty (obr\u00e1zek 2). Pom\u011br A <sub>260<\/sub> \/A <sub>280<\/sub> \u010dist\u00fdch roztok\u016f nukleov\u00fdch kyselin bude p\u0159ibli\u017en\u011b 1,8 &#8211; 2,0, zat\u00edmco pom\u011br A <sub>260<\/sub> \/A <sub>230<\/sub> bude typicky vykazovat hodnoty v rozmez\u00ed 2,0 &#8211; 2,5.<\/p>\n\n<figure class=\"wp-block-image size-large\"><img loading=\"lazy\" width=\"768\" height=\"445\" src=\"https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_Two_Applications_389bfe613c.jpg\" alt=\"\" class=\"wp-image-9791\" srcset=\"https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_Two_Applications_389bfe613c.jpg 768w, https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_Two_Applications_389bfe613c-690x400.jpg 690w, https:\/\/cotslab.com\/wp-content\/uploads\/2021\/10\/csm_Figure_Two_Applications_389bfe613c-600x348.jpg 600w\" sizes=\"(max-width: 768px) 100vw, 768px\" \/><figcaption>Obr\u00e1zek 2: Absorban\u010dn\u00ed spektra nukleov\u00fdch kyselin a mo\u017en\u00fdch kontaminant\u016f<\/figcaption><\/figure>\n\n<p>K ur\u010den\u00ed pozad\u00ed se pou\u017e\u00edv\u00e1 \u010dtvrt\u00e1 vlnov\u00e1 d\u00e9lka. M\u011b\u0159\u00ed se p\u0159i nebo nad 320 nm, proto\u017ee ani nukleov\u00e9 kyseliny ani organick\u00e9 kontaminanty neabsorbuj\u00ed sv\u011btlo p\u0159i t\u00e9to vlnov\u00e9 d\u00e9lce. Absorbance m\u011b\u0159en\u00e1 v tomto rozsahu m\u016f\u017ee indikovat p\u0159\u00edtomnost \u010d\u00e1stic nebo vzduchov\u00fdch bublin ve vzorku. Dokonce i rozmazan\u00e1 kyveta je schopna vyvolat \u010dten\u00ed na pozad\u00ed. Modern\u00ed fotometry nab\u00edzej\u00ed mo\u017enost aktivace funkce korekce pozad\u00ed, kter\u00e1 provede automatick\u00e9 ode\u010dten\u00ed jak\u00e9koli absorbance pozad\u00ed od v\u0161ech ostatn\u00edch nam\u011b\u0159en\u00fdch hodnot.<\/p>\n\n<p>Krom\u011b toho m\u016f\u017ee b\u00fdt zachyceno kompletn\u00ed spektrum vzorku nukleov\u00e9 kyseliny (typicky mezi 220 a 320 nm). Srovn\u00e1n\u00ed se spektrem z\u00edskan\u00fdm z \u010dist\u00e9ho roztoku nukleov\u00e9 kyseliny umo\u017en\u00ed detekci mo\u017en\u00fdch chyb b\u011bhem procesu m\u011b\u0159en\u00ed a tak\u00e9 p\u0159\u00edtomnost kontaminant\u016f ve vzorku.<\/p>\n\n<p><a href=\"http:\/\/handling-solutions.eppendorf.com\/\">Zdroj<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>M\u011b\u0159en\u00ed absorbance roztoku nukleov\u00e9 kyseliny pouze p\u0159i \u010dty\u0159ech vlnov\u00fdch d\u00e9lk\u00e1ch je dostate\u010dn\u00e9 pro stanoven\u00ed jej\u00ed koncentrace a pro z\u00edsk\u00e1n\u00ed n\u00e1hledu na jej\u00ed \u010distotu. Zdroj obr\u00e1zk\u016f: DRogatnev\/shutterstock.com Nukleov\u00e9 kyseliny jsou izolov\u00e1ny ze vzorku materi\u00e1lu, jako jsou bu\u0148ky, a n\u00e1sledn\u011b pou\u017eity v dal\u0161\u00edch laboratorn\u00edch experimentech. Pro tento \u00fa\u010del je vhodn\u00e9 stanovit koncentraci purifikovan\u00e9 DNA nebo RNA a [&#8230;]\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[483],"tags":[],"_links":{"self":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9783"}],"collection":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/comments?post=9783"}],"version-history":[{"count":1,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9783\/revisions"}],"predecessor-version":[{"id":9794,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/posts\/9783\/revisions\/9794"}],"wp:attachment":[{"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/media?parent=9783"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/categories?post=9783"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cotslab.com\/cs\/wp-json\/wp\/v2\/tags?post=9783"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}