{"id":9932,"date":"2022-02-28T12:10:54","date_gmt":"2022-02-28T12:10:54","guid":{"rendered":"https:\/\/cotslab.com\/fluaraiseacht-breis-luachmhar-leis-an-ionsuite\/"},"modified":"2022-02-28T12:11:00","modified_gmt":"2022-02-28T12:11:00","slug":"fluaraiseacht-breis-luachmhar-leis-an-ionsuite","status":"publish","type":"post","link":"https:\/\/cotslab.com\/ga\/fluaraiseacht-breis-luachmhar-leis-an-ionsuite\/","title":{"rendered":"Fluaraiseacht: Breis Luachmhar leis an Ions\u00faite?"},"content":{"rendered":"\n

D\u00e9antar aig\u00e9id n\u00faicl\u00e9acha agus pr\u00f3it\u00e9in\u00ed a chainn\u00edocht\u00fa go rialta tr\u00ed \u00fas\u00e1id a bhaint as speictreaf\u00f3taim\u00e9adracht UV-Vis; uaireanta, \u00e1fach, d’fh\u00e9adfadh braite tr\u00ed fhluaraiseacht a bheith n\u00edos fabhra\u00ed. Sa ch\u00e1s seo, n\u00ed aims\u00edtear m\u00f3il\u00edn\u00ed tr\u00ed ions\u00faiteacht, ach braitear iad ar bhealach ind\u00edreach – tr\u00ed chomhartha ruaime fluaraisigh. N\u00ed hamh\u00e1in go dtugann fluaraiseacht modh braite an-\u00edogair, ach ceada\u00edonn s\u00e9 anail\u00edt\u00ed aonair a thomhas go roghnach.<\/p>\n\n

C\u00e1s 1: Conas a rachaidh m\u00e9 ar aghaidh le r\u00e9itigh samplacha \u00edseal-ti\u00fachana?<\/strong><\/p>\n\n

N\u00edl an speictrophotim\u00e9adracht UV-Vis an-\u00edogair \u2013 chun sonra\u00ed beachta agus beachta a fh\u00e1il, n\u00edor cheart ligean do thi\u00fachan na sampla\u00ed titim faoi thairseach ti\u00fachana \u00e1irithe. Maidir le dsDNA, t\u00e1 an teorainn \u00edochtair seo thart ar 1 \u00b5g\/mL. Fi\u00fa mura bhfuil an teorainn braite ar an bhf\u00f3taim\u00e9adar sroichte, t\u00e1 tionchar an mh\u00edchruinnis tomhais sa raon seo suntasach, agus d’fh\u00e9adfadh go mbeadh \u00e9ags\u00falacht shuntasach sna luachanna mar thoradh air. T\u00e1 anail\u00eds fhluairmh\u00e9adrach, at\u00e1 1000-huaire n\u00edos \u00edogaire n\u00e1 an modh ions\u00fach\u00e1in, in ann r\u00e9itigh shamplacha de thi\u00fachan an-\u00edseal a chainn\u00edocht\u00fa go han-chruinn.<\/p>\n\n

C\u00e1s 2: Conas is f\u00e9idir liom cainn\u00edocht\u00fa cruinn a dh\u00e9anamh in ainneoin \u00e9illithe samplach?<\/strong><\/p>\n\n

Chomh maith le cainn\u00edocht\u00fa aig\u00e9id n\u00faicl\u00e9acha ag 260 nm, cuireann speictrophotim\u00e9adracht UV-Vis an rogha chun \u00edonacht samplach a chinneadh tr\u00ed thonnfhad breise (230 nm, 280 nm, 320 nm), n\u00f3 scanadh tonnfhad, faoi seach a \u00fas\u00e1id. Cead\u00f3idh anail\u00eds sonra\u00ed concl\u00faid\u00ed maidir le l\u00e1ithreacht substaint\u00ed amhail salann, pr\u00f3it\u00e9in\u00ed, n\u00f3 c\u00e1ithn\u00edn\u00ed soladacha. M\u00e1s rud \u00e9, ar an l\u00e1imh eile, go bhfuil na speictrim ions\u00fach\u00e1in r\u00f3-chos\u00fail, mar at\u00e1 i gc\u00e1s na n-aig\u00e9id n\u00faicl\u00e9acha \u00e9ags\u00fala, n\u00ed bheidh ions\u00faiteacht amh\u00e1in in ann idirdheal\u00fa a dh\u00e9anamh idir, mar shampla, RNA agus DNA. Sa ch\u00e1s seo, cead\u00f3idh tomhais fhluaraiseachta anail\u00edt\u00ed a chainn\u00edocht\u00fa go sonrach, gan cur isteach \u00f3 mh\u00f3il\u00edn\u00ed eile.<\/p>\n\n

C\u00e9n prionsabal ar a bhfuil an modh seo bunaithe?<\/strong><\/p>\n\n

Baineann tomhais fhluaraiseachta leas as an bhfeinim\u00e9an go bhfuil m\u00f3il\u00edn\u00ed \u00e1irithe in ann fuinneamh a ast\u00fa i bhfoirm solais. Chun bithmh\u00f3il\u00edn\u00ed amhail aig\u00e9id n\u00faicl\u00e9asacha agus pr\u00f3it\u00e9in\u00ed a bhrath, roghna\u00edtear dath fluaraiseacha a cheangail leis an anail\u00edt leis an saini\u00falacht is airde is f\u00e9idir. Is sampla clasaiceach \u00e9 nascadh an imoibr\u00ed PicoGreen \u00ae<\/sup> le dsDNA. Tar \u00e9is excitation an choimpl\u00e9asc ag baint \u00fas\u00e1ide as solas de thonnfhad ar leith (thart ar 480 nm do PicoGreen), scaoilfidh an fluorophore solas le fuinneamh n\u00edos \u00edsle, ie de thonnfhad n\u00edos faide (thart ar 520 nm do PicoGreen) (figi\u00far 1). D\u00e9antar an solas a thomhas mar fhluaraiseacht choibhneasta (RFU), \u00e1it a bhfuil d\u00e9ine an fhluaraiseachta comhr\u00e9ireach le ti\u00fachan an tsampla.<\/p>\n\n

\"\"
F\u00edor 1:A) Prionsabal tomhais fhluaraiseachta aig\u00e9id n\u00faicl\u00e9acha (ar chl\u00e9)B) T\u00e1 tonnfhad n\u00edos giorra n\u00e1 an solas astaithe (EM) (ar dheis) ag an solas a \u00fas\u00e1idtear chun an fluorophore (EX) a spreagadh<\/figcaption><\/figure><\/div>\n\n

C\u00e9 na h\u00e1bhair at\u00e1 ag teast\u00e1il don mhodh seo?<\/strong><\/p>\n\n

Baineann cainn\u00edocht\u00fa aig\u00e9id n\u00faicl\u00e9acha agus pr\u00f3it\u00e9in\u00ed leas as roghn\u00fa na ruaimeanna fluaraiseacha a cheangla\u00edonn a sprioc-mh\u00f3il\u00edn faoi seach le saini\u00falacht ard. Ina theannta sin, n\u00ed m\u00f3r caighde\u00e1n amh\u00e1in ar a laghad de thi\u00fachan aitheanta a bheith ar f\u00e1il. Teasta\u00edonn fluaraim\u00e9adar neamhsple\u00e1ch n\u00f3 mod\u00fal fluaraiseachta at\u00e1 comhth\u00e1ite laistigh d’ionstraim eile chun an fluaraf\u00f3r a ghr\u00edosadh agus chun d\u00e9ine fhluaraiseachta an tsolais astaithe a thomhas. Caithfidh an trealamh agus an dath fluaraiseacha a bheith comhoiri\u00fanach ionas go sol\u00e1thr\u00f3idh an fhoinse solais laistigh den uirlis an tonnfhad excitation riachtanach agus go tomhaiseann an brathad\u00f3ir an solas a asta\u00edtear. Ag brath ar an gcine\u00e1l ionstraime, t\u00e1 na r\u00e9itigh imoibr\u00ed suite i soith\u00ed imoibrithe le balla\u00ed tana\u00ed n\u00f3 i cuvettes. N\u00ed m\u00f3r comhoiri\u00fanacht leis an bhfeidhmchl\u00e1r sonrach maidir le feisti\u00fa, tr\u00e9dhearcacht, uathfhluaraiseacht agus toirt a chur san \u00e1ireamh.<\/p>\n\n

Conas a chuirtear an modh i gcr\u00edch?<\/strong><\/p>\n\n

Taispe\u00e1nann Figi\u00far 2 seicheamh tomhais fluairim\u00e9adracha. Cuirtear t\u00fas le hullmh\u00fa an bh\u00e1na, na gcaighde\u00e1n agus an tsampla tr\u00edd an dath fluaraiseachta a chur leis, agus tr\u00e9imhse goir ghearr ina dhiaidh sin. Ar dt\u00fas, d\u00e9antar caighde\u00e1n amh\u00e1in n\u00f3 n\u00edos m\u00f3 de chomhchruinnithe aitheanta a thomhas chun an cuar caighde\u00e1nach a ghini\u00faint. D\u00e9antar ti\u00fachan an tsampla a chinneadh ina dhiaidh sin sa dara c\u00e9im, ina nd\u00e9antar d\u00e9ine fluaraiseachta tomhaiste na sampla\u00ed a mheas i gcompar\u00e1id leis an gcuar caighde\u00e1nach.<\/p>\n\n

\"\"
Fluaraiseacht sreabhadh oibre<\/figcaption><\/figure><\/div>\n\n

Fluaraiseacht \u2013 modh roghnaithe?<\/strong><\/p>\n\n

Fi\u00fa m\u00e1 t\u00e1 bunt\u00e1ist\u00ed ag baint le fluairim\u00e9adracht i gc\u00e1sanna \u00e1irithe, leanann speictrophotim\u00e9adracht clasaiceach UV-Vis de bheith mar an modh caighde\u00e1nach maidir le haig\u00e9id n\u00faicl\u00e9acha agus pr\u00f3it\u00e9in\u00ed a chainn\u00edocht\u00fa. Ceada\u00edonn speictrophotim\u00e9adracht \u00edonacht an tsampla a fh\u00edor\u00fa, agus is f\u00e9idir anail\u00eds dh\u00edreach a dh\u00e9anamh ar r\u00e9itigh an-chomhchruinnithe. Ag brath ar ch\u00e1il\u00edocht an tsampla, chomh maith le riachtanais iarratais iartheachtacha, b’fh\u00e9idir go mbeadh s\u00e9 inmholta an d\u00e1 theicn\u00edc a chomhcheangal. <\/p>\n\n

Is tr\u00e1dmharc cl\u00e1raithe \u00e9 PicoGreen \u00ae<\/sup> de Molecular Probes, Inc. Corporation, Eugene, N\u00d3, SAM. Is tr\u00e1dmharcanna cl\u00e1raithe de chuid Eppendorf AG, an Ghearm\u00e1in iad Eppendorf \u00ae<\/sup> , an Eppendorf Brand Design, Eppendorf BioSpectrometer \u00ae<\/sup> , Uvette \u00ae<\/sup> agus Eppendorf \u00b5Cuvette \u00ae \u00ae<\/sup> . Gach ceart ar cosaint lena n-\u00e1ir\u00edtear grafaic agus \u00edomh\u00e1nna. C\u00f3ipcheart \u00a9 2019 Eppendorf AG.<\/em><\/p>\n\n

Foinse<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

D\u00e9antar aig\u00e9id n\u00faicl\u00e9acha agus pr\u00f3it\u00e9in\u00ed a chainn\u00edocht\u00fa go rialta tr\u00ed \u00fas\u00e1id a bhaint as speictreaf\u00f3taim\u00e9adracht UV-Vis; uaireanta, \u00e1fach, d’fh\u00e9adfadh braite tr\u00ed fhluaraiseacht a bheith n\u00edos fabhra\u00ed. Sa ch\u00e1s seo, n\u00ed aims\u00edtear m\u00f3il\u00edn\u00ed tr\u00ed ions\u00faiteacht, ach braitear iad ar bhealach ind\u00edreach – tr\u00ed chomhartha ruaime fluaraisigh. N\u00ed hamh\u00e1in go dtugann fluaraiseacht modh braite an-\u00edogair, ach ceada\u00edonn […]\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[489],"tags":[],"_links":{"self":[{"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/posts\/9932"}],"collection":[{"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/comments?post=9932"}],"version-history":[{"count":1,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/posts\/9932\/revisions"}],"predecessor-version":[{"id":9940,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/posts\/9932\/revisions\/9940"}],"wp:attachment":[{"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/media?parent=9932"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/categories?post=9932"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cotslab.com\/ga\/wp-json\/wp\/v2\/tags?post=9932"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}