Ukuze uthole ukusabela okuphumelelayo kwe-amplification, kuyadingeka ukuthi izingxenye zokusabela ngazinye zibe khona ekugxiliseni okufanele ekulungiseleleni ngakunye.Ngaphezu kwalokho, kubalulekile ukuthi kungabikho ukungcola okwenzekayo.
Ikakhulukazi lapho ukusabela okuningi kufanele kusethwe, kuye kwasungulwa ukuze kulungiswe lokho okubizwa ngokuthi i-master mix esikhundleni sokufakela i-reagent ngayinye ngokuhlukana emkhunjini ngamunye.Amamiksi alungiselelwe kusengaphambili ayatholakala ngokwentengiso, lapho kuphela izingxenye ezithile zesampula (i-primer) namanzi engezwayo.Kungenjalo, i-master mix ingalungiswa uwena.Kuzo zombili izinhlobo, ingxube isatshalaliswa emkhunjini ngamunye we-PCR ngaphandle kwesifanekiso futhi isampula ye-DNA ngayinye yengezwa ngokuhlukile ekugcineni.
Ukusebenzisa i-master mix kunezinzuzo eziningana: Okokuqala, inani lezinyathelo zepayipi elilodwa liyancipha.Ngale ndlela, kokubili ingozi yamaphutha omsebenzisi ngesikhathi sokufaka amapayipi kanye nengozi yokungcola iyancishiswa futhi, yiqiniso, isikhathi sigcinwa.Eqinisweni, ukunemba kwamapayipi nakho kuphezulu, njengoba amavolumu amakhulu athathiwe.Lokhu kulula ukukuqonda lapho uhlola idatha yobuchwepheshe yama-pipette: Uma umthamo wethamo lincane, ukuphambuka kungaba phezulu.Iqiniso lokuthi wonke amalungiselelo avela emkhunjini ofanayo anomphumela omuhle ku-homogeneity (uma ixutshwe kahle).Lokhu futhi kuthuthukisa ukukhiqizwa kabusha kokuhlolwa.
Lapho ulungiselela i-master mix, okungenani i-10 % ivolumu eyengeziwe kufanele yengezwe (isb uma amalungiselelo ayi-10 edingekayo, bala ngesisekelo se-11), ukuze ngisho nomkhumbi wokugcina ugcwale kahle.Ngale ndlela, ukunemba kwamapayipi (okuncane), kanye nomthelela wokulahleka kwesampula lapho ukufakwa kwezixazululo eziqukethe okokuhlanza kunganxeshezelwa.Izihlanzi ziqukethwe kuzixazululo ze-enzyme ezifana nama-polymerase nezingxube eziyinhloko, okubangela ukwakheka kwegwebu nezinsalela endaweni engaphakathi evamile.amathiphu we-pipette.
Ngokuya ngokusetshenziswa kanye nohlobo loketshezi oluzokhishwa, indlela efanele yokufaka amapayipi (1) kufanele kukhethwe futhi kukhethwe okokusebenza okufanele.Ukuze uthole izixazululo eziqukethe ama-detergents, uhlelo lokufuduka oluqondile noma lokho okubizwa ngokuthi "ukugcinwa okuphansi" amathiphu e-pipette njengenye indlela yamapayipi e-air-cushion anconywa.Umphumela weIthiphu ye-ACE PIPETTEisekelwe endaweni ikakhulukazi ye-hydrophobic.Uketshezi oluqukethe okokuhlanza alushiyi ifilimu eyinsalela ngaphakathi nangaphandle, ukuze ukulahlekelwa kwesisombululo kuncishiswe.
Ngaphandle komthamo oqondile wazo zonke izingxenye, kubalulekile futhi ukuthi kungabikho ukungcoliswa kwamalungiselelo okwenzekayo.Akwanele ukusebenzisa izinto ezisetshenziswayo zokuhlanzeka okuphezulu, ngoba inqubo yokufaka ipayipi ku-pipette ye-air cushion ingakhiqiza ama-aerosol ahlala ku-pipette.I-DNA engase iqukethwe ku-aerosol ingadluliswa isuka kusampula eyodwa iye kwelandelayo esinyathelweni samapayipi esilandelayo futhi ngaleyo ndlela iholele ekungcoleni.Izinhlelo eziqondile zokususa ezishiwo ngenhla nazo zingayinciphisa le ngozi.Kuma-pipette e-air-cushion kunengqondo ukusebenzisa amathiphu okuhlunga ukuvikela ikhoni ye-pipette ngokugcina ama-splashes, ama-aerosol, nama-biomolecules.
Isikhathi sokuthumela: Dec-06-2022