Ngisho noma ngokombono, i-molecule eyodwa yesifanekiso izokwanela, amanani amakhulu kakhulu e-DNA ngokuvamile asetshenziselwa i-PCR yakudala, isibonelo, kufika ku-1 µg we-DNA yezilwane ezincelisayo kanye nengxenye encane efinyelela ku-1 pg ye-plasmid DNA.Inani eliphelele lincike kakhulu enanini lamakhophi ochungechunge oluqondiwe, kanye nokuba yinkimbinkimbi kwalo.
Uma isifanekiso esincane kakhulu sisetshenziswa, ukwanda okuhambisanayo kwenani lemijikelezo yokukhulisa izwi kuyodingeka ukuze kutholwe inani elanele lomkhiqizo.I-Taq polymerase esetshenziselwa ukuhlola okuningi kwe-PCR ayinawo umsebenzi wokulungisa (3′-5′ umsebenzi we-exonuclease);ngakho, amaphutha enzeka ngesikhathi sokukhulisa awakwazi ukulungiswa.Lapho inani lemijikelezo liphakeme, kuzokwanda ukwanda komkhiqizo onephutha.Uma, ngakolunye uhlangothi, inani lesifanekiso liphezulu kakhulu, amathuba okuba ama-primer axhunywe kokunye (hhayi amaphesenti ayikhulu okuncoma) ukulandelana, kanye nokwakhiwa kwama-primer dimers, azokhula, okuzoholela ekwandiseni ngemikhiqizo.Ezimweni eziningi, i-DNA ihlukanisiwe kumasiko amaseli noma kuma-microorganisms bese isetshenziswa njengesifanekiso se-PCR.Ukulandela ukuhlanzwa, kuyadingeka ukunquma ukugxila kwe-DNA ukuze ukwazi ukuchaza ivolumu edingekayo ekusethweni kwe-PCR.Nakuba i-agarose gel electrophoresis ingase inikeze isilinganiso, le ndlela ayinembile neze.I-spectrophotometry ye-UV-Vis isungulwe njengezinga legolide lokulinganisa ama-nucleic acid;le ndlela eqondile futhi ngakho-ke elula futhi esheshayo ikala ukumuncwa kwesampula ku-260 nm, futhi ukugxila kubalwa ngosizo lwesici sokuguqula.
Uma ukugxila kwe-DNA kuphansi kakhulu, nokho (< 1 µg/mL dsDNA), noma uma kungcoliswe izinto ezimunca futhi ku-260 nm range (isb. i-RNA, amaprotheni, usawoti), le ndlela izofinyelela ukulinganiselwa kwayo.Esimeni sokugxilisa okuphansi kakhulu, ukufundwa kuzobe kunganembile kakhulu ukuthi kungasetshenziswa, futhi ukungcoliswa kuzoholela (ngezinye izikhathi kukhulu) ekulinganisweni ngokweqile kwevelu yangempela.Kulokhu, ukulinganisa kwe-fluorescence kungase kubonise enye indlela.Le nqubo isuselwe ekusetshenzisweni kodayi we-fluorescent obophezela ngokuqondile ku-dsDNA kuphela inkimbinkimbi ehlanganisa i-nucleic acid nodayi ujatshuliswa ukukhanya, futhi ngemva kwalokho izokhipha ukukhanya kobude beza obuphakeme kancane.Lapha, ukushuba kwesignali ye-fluorescent ilingana nenani le-DNA, futhi ukuze kunqunywe ukugxilisa ingqondo kuyahlolwa ngokuhlobene nejika elijwayelekile.Izinzuzo zale ndlela zincike ekucacisweni kwebhondi, okungabandakanyi amathonya angaphandle alethwa ukungcola, kanye nasemandleni okuba umphumela wokuthola ukugxila okuphansi kakhulu kwe-DNA.Ukufaneleka kwanoma iyiphi indlela kuncike kakhulu ekugxilweni kwesampula nokuhlanzeka;ezimweni eziningi kungase kube kuhle ukusebenzisa izindlela zombili ngokuhambisana.
Isikhathi sokuthumela: Nov-30-2022