Noma ngombono, i-molecule eyodwa yethempeli ibingase inele, inani elikhudlwana le-DNA ngokuvamile lisetshenziselwa i-pcr yasendulo, isibonelo, kuze kube yi-1 μg ye-genomic mammalian DNA futhi kancane njenge-1 pg ye-plasmid DNA. Inani elifanele lincike kakhulu kwinani lamakhophi okulandelana okuqondiwe, kanye nakwinkimbinkimbi yawo.
Uma kusetshenziswa ithempulethi encane kakhulu, ukukhuphuka okuhambisanayo kwenombolo yemijikelezo yokukhulisa kuzodingeka ukuthola inani elanele lomkhiqizo. I-taq polymerase esetshenziselwa izivivinyo eziningi ze-PCR ayifaki umsebenzi wokulungiswa (3'-5 'EXOCUCLESE umsebenzi); Ngakho-ke, kwenzeka amaphutha ngesikhathi sokukhulisa ngeke alungiswe. Lapho inani lemijikelezo, okuzokwanda kakhulu okuzothuthukiswa komkhiqizo onephutha. Uma, ngakolunye uhlangothi, inani lethempulethi liphezulu kakhulu, amathuba okuba ama-primers anveling kwezinye izindlela (hhayi eziyikhulu amaphesenti ancompyration), kanye nokwakhiwa kwezindawo zokuphrinta, kuzokhuphuka, okuzoholela ekulweni kwe-Primer. ngemikhiqizo. Ezimweni eziningi, i-DNA ihlukaniswe namasiko weseli noma ama-microorganisms futhi asetshenziswa kamuva njengethempulethi ye-PCR. Ngemuva kokuhlanzwa, kuyadingeka ukuthola ukugcwaliseka kwe-DNA ukukwazi ukuchaza ivolumu edingekayo e-PCC Set. Ngenkathi i-agarose gel electrophoresis ingahle inikeze ukulinganisa, le ndlela ayinayo enembile. I-UV-Vis spectrophotometry isungulwe njengezinga legolide lokulinganisa kwama-nucleic acid; Lokhu kuqondile futhi ngakho-ke indlela elula futhi esheshayo yokumunca isampula ku-260 NM, kanye nokuhlushwa kubalwa ngosizo lwento yokuguqula.
Uma ukugxila kwe-DNA kuphansi kakhulu, noma kunjalo (<1 μg / ml DSDNA), noma uma kungcoliswe ngezinto ezingenisa nobubanzi be-260 nm (isib. I-RNA, le ndlela izofinyelela ukulinganiselwa kwayo. Endabeni yokugxila okuphansi kakhulu, ukufundwa maduze nje kuzolunga kakhulu ukuba kube ukusetshenziswa, kanti ama-contacontations azoholela ekulweni kwenani langempela. Kulokhu, ubungako usebenzisa i-fluorescence bungalekela enye indlela. Le ndlela isuselwa ekusetshenzisweni udayi we-fluorescent ohlanganisa ngqo i-DSDNA kuphela i-nucleic acid nodayi ebabazekayo kujabulise ukukhanya, futhi kuzolandela ukukhanya kwe-wavelength ephakeme kancane kancane. Lapha, ukuqina kwesiginali ye-fluorescent kulingana nenani le-DNA, kanye nokunquma ukugxila kuhlolwe maqondana nejika elijwayelekile. Izinzuzo zale ndlela ziphumule ngokucaciswa kwesibopho, ezingafakwanga amathonya angaphandle ezethulwe ngokungcoliswa, kanye nakwikhono okuholela ekutholeni ukugxila okuphansi kakhulu kwe-DNA. Ukufaneleka kwendlela noma iyiphi indlela kuncike kakhulu ekuhlungenwe kwampunga nobumsulwa; Ezimweni eziningi kungahle kube lula ukusebenzisa zombili izindlela ngokufana.
Isikhathi sePosi: Nov-30-2022