Ukumiswa kwezinqubo kuhlanganisa ukuthuthukiswa kwazo kanye nokusungulwa nokuvumelanisa okulandelayo, okuvumela ukusebenza okuphezulu kwesikhathi eside - ngaphandle komsebenzisi. Ukulinganisa kuqinisekisa imiphumela yekhwalithi ephezulu, kanye nokuphindaphindeka kwayo nokuqhathaniswa.
Umgomo we-PCR (yakudala) ukukhiqiza umphumela onokwethenjelwa nophindaphindeka. Kwezinhlelo zokusebenza ezithile, isivuno se-PCR umkhiqizokuyafaneleka futhi. Kulokhu kusabela, kufanele kuqashelwe ukuze kuqinisekiswe ukuthi amasampula awekho engcupheni nokuthi ukuhamba komsebenzi kwe-PCR kuhlala kuzinzile. Ikakhulukazi, lokhu kuhumushela ekunciphiseni ukwethulwa kokungcola okungaholela emiphumeleni engemihle noma engamanga engemihle noma kuvimbele ukusabela kwe-PCR. Ngaphezu kwalokho, izimo zokusabela kufanele zifane ngangokunokwenzeka kusampula ngayinye ngesikhathi sokugijima futhi zidluliselwe ekuphenduleni okulandelayo (kwendlela efanayo). Lokhu kubhekisela ekwakhiweni kokusabela kanye nohlobo lokulawula izinga lokushisa kumjikelezo. Amaphutha omsebenzisi, vele, kufanele agwenywe ngangokunokwenzeka.
Ngezansi, sizokhombisa izinselelo okuhlangatshezwana nazo ngesikhathi sokulungiselela kanye naso sonke isikhathi sokuqhutshwa kwe-PCR - kanye nezindlela zezixazululo ezikhona maqondana namathuluzi nezinto ezisetshenziswayo ezisetshenziselwa ukumiswa kokuhamba komsebenzi kwe-PCR.
Ukulungiselela ukusabela
Ukusatshalaliswa kwezingxenye zokusabela emikhunjini ye-PCR, noma amapuleti, ngokulandelana, kuhlanganisa izinselele eziningi okufanele zinqotshwe:
Izimo zokusabela
Umthamo onembile futhi onembe wezingxenye ngazinye kudingekile uma uhloselwe izimo zokusabela ezifanayo kakhulu ngangokunokwenzeka. Ngaphezu kobuchwepheshe obuhle bepayipi, ukukhetha ithuluzi elifanele kubalulekile. Amamiksi e-PCR master ngokuvamile aqukethe izinto ezandisa i-viscosity noma ezikhiqiza igwebu. Ngesikhathi se-pipetting, lezi ziholela ekumanziseni okukhulu kwe-amathiphu we-pipette, ngaleyo ndlela kunciphisa ukunemba kwamapayipi. Ukusetshenziswa kwezinhlelo eziqondile zokusabalalisa noma ezinye izeluleko ze-pipette ezingakwazi ukumanzisa zingathuthukisa ukunemba nokunemba kwenqubo yepayipi.
Ukungcola
Phakathi nenqubo yokukhipha, ama-aerosols akhiqizwa okuthi, uma evunyelwe ukuba afinyelele ngaphakathi kwe-pipette, angakwazi ukungcolisa enye isampula ngesikhathi sesinyathelo esilandelayo sepayipi. Lokhu kungavinjelwa ngokusebenzisa amathiphu okuhlunga noma amasistimu okuthutha aqondile.
Izinto ezisetshenziswayo njengeamathiphu, imikhumbi namapuleti asetshenziswa ekuhambeni komsebenzi kwe-PCR akumele aqukathe izinto ezifaka engozini isampula noma ezikhohlisa umphumela. Lokhu kufaka phakathi i-DNA, i-DNases, i-RNases kanye ne-PCR inhibitors, kanye nezingxenye ezingase ziphume entweni ngesikhathi sokusabela - izinto ezaziwa ngokuthi ezivuzayo.
Iphutha lomsebenzisi
Uma amasampula engeziwe ecutshungulwa, iba nkulu ingozi yamaphutha. Kungenzeka kalula ukuthi isampula lifakwe ngepayipi emkhunjini ongalungile noma emthonjeni ongalungile. Le ngozi ingancishiswa kakhulu ngokubonakala kalula kwemithombo. Ngokuzenzakalela kwezinyathelo zokukhipha, “isici somuntu”, okungukuthi, amaphutha nokuhluka okuhlobene nomsebenzisi, kuyancishiswa, ngaleyo ndlela kwandise ukuphindaphindeka, ikakhulukazi esimweni samavolumu amancane okusabela. Lokhu kudinga amapuleti okuzinza kwe-dimensional eyanele ukuze aqashwe endaweni yokusebenza. Amabhakhodi anamathiselwe ahlinzeka ngokufundeka komshini okwengeziwe, okwenza ukulandelela isampula kube lula kuyo yonke inqubo.
Uhlelo lwe-thermocycler
Ukuhlela ithuluzi kungase kudle isikhathi futhi kube namaphutha. Izici ezihlukene zomjikelezo oshisayo we-PCR zisebenza ndawonye ukwenza lula lesi sinyathelo senqubo futhi, okubaluleke kakhulu, ukulenza liphephe:
Ukusebenza okulula nokuqondisa okuhle komsebenzisi kuyisisekelo sokuhlela okusebenzayo. Ngokwakhela phezu kwalesi sisekelo, ukuphathwa komsebenzisi ovikelwe ngephasiwedi kuzovimbela izinhlelo zomuntu siqu ukuthi zingashintshwa abanye abasebenzisi. Uma amabhayisikili amaningi (ohlobo olufanayo) esetshenziswa, kunenzuzo uma uhlelo lungadluliswa ngokuqondile lusuka kwelinye ithuluzi luye kwelinye nge-USB noma ngoxhumano. Isofthiwe yekhompiyutha inika amandla ukuphathwa okumaphakathi nokuvikelekile kwezinhlelo, amalungelo abasebenzisi kanye nemibhalo kukhompuyutha.
Ukusebenza kwe-PCR
Ngesikhathi sokugijima, i-DNA ikhuliswa emkhunjini wokusabela, lapho isampula ngayinye kufanele ibe ngaphansi kwezimo ezifanayo, ezingaguquki zokusabela. Izici ezilandelayo zibalulekile kule nqubo:
Ukulawula izinga lokushisa
Ukunemba okuhle kakhulu ekulawuleni izinga lokushisa kanye ne-homogeneity yebhulokhi yomjikelezo kuyisisekelo sokumisa ngisho nezinga lokushisa kwawo wonke amasampuli. Izinga eliphezulu lezinto zokushisa nokupholisa (izakhi ze-peltier), kanye nendlela lezi ezixhunywe ngayo ku-block, yizici ezinqumayo ezizonquma ingozi yokungafani kwezinga lokushisa eyaziwa ngokuthi "umphumela onqenqemeni"
Ukuhwamuka
Ukugxila kwezingxenye zokusabela ngakunye akufanele kuguquke phakathi nesikhathi sokusabela ngenxa yokuhwamuka. Kungenjalo, kungenzeka ukuthi kuncane kakhuluPCR umkhiqizoingase yenziwe, noma ingabi khona nhlobo. Ngakho-ke kubalulekile ukunciphisa ukuhwamuka ngokuqinisekisa uphawu oluvikelekile. Kulesi simo, isivalo esishisayo se-thermocycler kanye nophawu lomkhumbi lusebenza ngesandla. Izinketho zokuvala ezihlukene ziyatholakalaAmapuleti e-PCR (isixhumanisi: Isihloko sokuvala), lapho uphawu oluhle kakhulu lutholakala ngokuvala ukushisa. Okunye ukuvala kungase kufane, inqobo nje uma ukucindezela kokuxhumana kwesivalo somjikelezo kungalungiswa kuphawu olukhethiwe.
Ukumiswa kwenqubo kusendaweni ukuze kuvikelwe imiphumela enembayo nengaphinda ikhiqizeke esikhathini eside. Lokhu kuhlanganisa ukugcinwa njalo kwezinto zokusebenza ukuze kuqinisekiswe ukuthi zihlala zisesimweni esihle sokusebenza. Konke okusebenzisekayo kufanele kube ngokwekhwalithi ephezulu ngokungaguquki kuzo zonke izindawo ezikhiqizwayo, futhi ukutholakala kwazo okuthembekile kufanele kuqinisekiswe.
Isikhathi sokuthumela: Nov-29-2022