Kukho amabala amaninzi ahlukeneyo angasetyenziselwa ukujonga kunye nokufotoza i- DNA emva kokuba impahla ihlukaniswe yi-gel electrophoresis. Phakathi kwezinto ezininzi ezikhethiweyo, ezi ntlobo ezinhlanu ziqala ngokuqhelekileyo kwi-Ethidium bromide, eyona nto isetyenziswa kakhulu. Xa usebenza nale nkqubo, kubalulekile ukuba ungabi nje kuphela ulwahluko phakathi kwemimandla kodwa ingozi yempilo.
01 Ethidium Bromide
I-ethidium bromide mhlawumbi idayi eyaziwayo kakhulu esetyenziselwa ukujonga i-DNA. Le dayi ingasetyenziselwa kumxube we-gel, kwi- taprophoresis buffer okanye ukucosa i-gel emva kokuba iqhutywe. Iimleksi zedayi zihambelane ne-DNA kunye ne-fluoresce ngaphantsi kokukhanya kwe-UV, ekuboniseni ukuba ziphi iindawo ezikhoyo kwi-gel. Nangona kunjalo, ukuphazamiseka kukuba i-ethidium bromide i-carcinogen, kwaye kufuneka iphathwe ngokunyamekela. Ngenxa yeengozi zempilo zokusebenza nale gel, ezinye iindlela zifunyenwe. 02 SYBR Gold
I-SYBR Idayi yegolide ingasetyenziselwa ukuhlamba i-DNA okanye i-DNA eyodwa-okanye i-RNA. I-SYBR Igolide yayingenye yezindlela zokuqala ezibhekiselele kwi-ethidium bromide ukubetha kwiimarike kwaye ithathwa njengentliziyo engathandeki kune-ethidium bromide. Idayi ibonisa i-UV-fluorescence enkulu ye-1000 ngokuphindaphindiweyo xa iboshwe kwi-nucleic acid. Emva koko kufinyelela kwipesenti eziphezulu kunye neephakamileyo ze-agarose gels kwaye zingasetyenziswa kwi-geldedede ze-gel. Ngenxa yokuba i-fluorescence ye-molecule engafihliyo iphantsi kakhulu, ukufunyanwa akufuneki. Umnini-mvume weMelecular Probes (kwakhona ukususela ekuqalisweni kwe-SYBR Gold) iphuhliswe kwaye ithengiswa i-SYBR Safe kunye ne-SYBR Green, eyindlela ekhuselekileyo yokubhekiselele kwi-ethidium bromide.
03 SYBR Green
I-SYBR Green I and II stains (kwakhona, ezithengiswa yiMelecular Probes) zilungiselelwe ngeenjongo ezahlukeneyo. Ngenxa yokuba zibophelela kwi-DNA, ziyakucingelwa ukuba zinokuthi ziphathwe ngokutsha kwaye ngenxa yoko, zifanele ziphathwe ngononophelo. I-SYBR I-Green iyakhathazeka kakhulu ukusebenzisa i-DNA ephindwe kabini, ngelixa i-SYBR Green II, ngakolunye uhlangothi, iyona nto ingcono ukusebenzisa i-DNA okanye i-RNA enye. Njengothutho oludumile lwe-Ethidium Bromide, ezi ndawo zibuhlungu kakhulu ziphazamisekile phantsi kwe-UV ukukhanya. 04 SYBR Safe
I-SYBR Safe iphethwe ukuba ibe yindlela ekhuselekileyo kwi-Ethidium Bromide kunye namanye ama-SYBR. Ayithathwa njengenkunkuma enobungozi kwaye ngokubanzi iyakuthi ilahlwe ngamashishini ahlaziyekileyo (oko kukuthi i-drain drain), kuba uvavanyo lwezobisi luchaza ukuba akukho buhlungu obuyingozi. Ukuvavanya kubonisa nokuba akukho ncinane okanye akukho nhlobo ye-genotoxicity kwiiseli zaseSiriya ze-Hamster Embryo (SHE), i-lymphocyte yabantu, iiseli ze-lymphoma ze-mouse kunye nokuhlolwa kwe-AMES. I-stain ingasetyenziswa nge-transilluminator eluhlaza okwesibhakabhaka ebangela ukuba umonakalo omkhulu kwi-DNA iboniswe kwaye unikezela ngokufanelekileyo ukusebenza kwe-cloning. 05 Eva Green
I-Eva Green yidayi ye-fluorescent eluhlaza efunyenwe ukuvimbela i- PCR ukuya ngaphantsi kunezinye idayi. Oku kwenza ukuba luncedo kakhulu kwizicelo ezinjenge-PCR yangempela-time. Kwakhona kukhethwa kakuhle ukuba usebenzisa i-low-melting point gels yokufumana i-DNA. Lizinzile kakhulu kumaqondo aphezulu kwaye i-fluorescence ephantsi kakhulu, kodwa i-fluorescent kakhulu xa iboshwe kwi-DNA. I-Eva Green iye yaboniswa ukuba iphantsi kakhulu okanye akukho cytotoxicity okanye mutagenicity.