Ukubekwa emgangathweni kweenkqubo kubandakanya ukulungelelaniswa kwazo kunye nokusekwa okulandelayo kunye nokulungelelaniswa, ukuvumela ukusebenza okugqibeleleyo kwexesha elide - ngaphandle komsebenzisi. Ukumiselwa komgangatho kuqinisekisa iziphumo ezikumgangatho ophezulu, kunye nokuveliswa kwazo kunye nokuthelekisa.
Injongo ye-PCR ye-(classic) sisizukulwana sesiphumo esithembekileyo kunye nokuphindaphinda. Kwizicelo ezithile, isivuno seImveliso yePCRikwabalulekile. Kwezi mpendulo, kufuneka kuthathelwe ingqalelo ukuqinisekisa ukuba iisampuli azichaphazeleki kwaye ukuhamba komsebenzi kwe-PCR kuhlala kuzinzile. Ngokukodwa, oku kuguqulela ekunciphiseni ukuqaliswa kosulelo olunokuthi lukhokelele kwiziphumo ezingezizo zobuxoki okanye ezingezizo ezingalunganga okanye zithintele ukusabela kwePCR. Ngaphaya koko, iimeko zokusabela kufuneka zifane kangangoko kwisampulu yomntu ngamnye ngaphakathi kokubaleka kwaye kwakhona idluliselwe kwiimpendulo ezilandelayo (zendlela enye). Oku kubhekiselele ekubunjweni kweempendulo kunye nohlobo lolawulo lobushushu kwi-cycler. Iimpazamo zabasebenzisi, ngokuqinisekileyo, kufuneka ziphetshwe kangangoko kunokwenzeka.
Apha ngezantsi, siza kubonisa imingeni esidibana nayo ngexesha lokulungiselela kunye nakulo lonke ixesha lokuqhutywa kwe-PCR - kunye neendlela zokusombulula ezikhoyo ngokubhekiselele kwizixhobo kunye nezinto ezisetyenziswayo ezisetyenziselwa ukulinganisa ukuhamba komsebenzi we-PCR.
Ukulungiselela ukusabela
Ukuhanjiswa kwezinto zokusabela kwi-PCR-imikhumbi, okanye iipleyiti, ngokulandelelanayo, kubandakanya imiceli mngeni emininzi ekufuneka yoyiswe:
Iimeko zokusabela
Eyona nto ichanekileyo kunye nechanekileyo yokudosa yamacandelo ngamanye ayimfuneko xa kujoliswa kwezona meko zokusabela zifanayo kunokwenzeka. Ukongeza kwindlela efanelekileyo yokufaka imibhobho, ukukhetha isixhobo esifanelekileyo kubalulekile. I-PCR master-mix ihlala iqulethe izinto ezandisa i-viscosity okanye i-foam. Ngexesha lenkqubo yokufaka imibhobho, ezi zikhokelela ekumanziseni okukhuluiingcebiso zepipette, ngaloo ndlela ukunciphisa ukuchaneka kwemibhobho. Ukusetyenziswa kweenkqubo zokukhupha ngokuthe ngqo okanye ezinye iingcebiso ze-pipette ezingaphantsi kwe-wetting zingaphucula ukuchaneka nokuchaneka kwenkqubo yepayipi.
Usulelo
Ngethuba lenkqubo yokusabalalisa, i-aerosols yenziwa ukuba, ukuba ivunyelwe ukuba ifikelele ngaphakathi kwi-pipette, inokuthi ingcolise enye isampuli ngexesha lesinyathelo esilandelayo sombhobho. Oku kunokuthintelwa ngokusebenzisa iingcebiso zokucoca okanye iinkqubo zokufuduswa ngokuthe ngqo.
Izinto ezisetyenziswayo ezifanaiingcebiso, iinqanawa kunye neepleyiti ezisetyenziswa kwi-PCR workflow kufuneka zingaqulathi izinto eziphazamisa isampuli okanye ziphosakele umphumo. Ezi ziquka i-DNA, i-DNases, i-RNases kunye ne-PCR inhibitors, kunye namacandelo anokuthi aphume kwizinto eziphathekayo ngexesha lokuphendula-izinto ezibizwa ngokuba yi-leachables.
Impazamo yomsebenzisi
Iisampulu ezininzi zicutshungulwa, kokukhona umngcipheko wempazamo uphezulu. Kwenzeka lula ukuba isampuli ifakwe kwisitya esingalunganga okanye iqula elingalunganga. Lo mngcipheko unokuncitshiswa kakhulu ngokuphawulwa ngokulula kwamaqula. Ngokuzenzekela kwamanyathelo okuhambisa, "into yomntu", oko kukuthi, iimpazamo kunye nokwahluka okunxulumene nomsebenzisi, kuncitshiswa, ngaloo ndlela kwandisa ukuveliswa kwakhona, ngakumbi kwimeko yemilinganiselo encinci yokusabela. Oku kufuna ukuba iipleyiti zozinzo olwaneleyo lwe-dimensional ziqeshwe kwindawo yokusebenzela. Iibhakhowudi ezincanyathiselweyo zibonelela ngokufundeka komatshini okongezelelweyo, okwenza kube lula ukulandelwa kwesampulu kuyo yonke inkqubo.
Ukucwangcisa i-thermocycler
Ukucwangcisa isixhobo kunokungqina ukuba kudla ixesha kunye nempazamo. Iimpawu ezahlukeneyo ze-PCR ze-thermal cycler zisebenza kunye ukwenza lula le nkqubo kwaye, okona kubaluleke kakhulu, ukuyenza ikhuseleke:
Ukusebenza ngokulula kunye nesikhokelo esilungileyo somsebenzisi sisiseko senkqubo esebenzayo. Ukwakha phezu kwesi siseko, ulawulo lomsebenzisi olukhuselweyo luya kuthintela iinkqubo zomntu ukuba zingatshintshwa ngabanye abasebenzisi. Ukuba iibhayisekile ezininzi (zohlobo olufanayo) ziyasebenza, kuyanceda ukuba inkqubo inokukhutshelwa ngokuthe ngqo ukusuka kwesinye isixhobo ukuya kwesinye nge-USB okanye uqhagamshelo. Isoftware yekhompyuter yenza ulawulo oluphakathi nolukhuselekileyo lweenkqubo, amalungelo omsebenzisi kunye namaxwebhu ekhompyuter.
Ukusebenza kwePCR
Ngethuba lokuqhuba, i-DNA ikhuliswe kwisitya sokuphendula, apho isampuli nganye kufuneka ifakwe kwiimeko ezifanayo, ezihambelanayo zokuphendula. Le miba ilandelayo ibalulekile kwinkqubo:
Ulawulo lobushushu
Ukuchaneka okugqwesileyo kulawulo lobushushu kunye ne-homogeneity yebhloko ye-cycler sisiseko somgangatho wobushushu bazo zonke iisampuli. Umgangatho ophezulu wezinto zokufudumeza kunye nokupholisa (izinto ze-peltier), kunye nendlela ezi zixhunywe ngayo kwibhloko, zizinto ezithatha isigqibo eziza kugqiba umngcipheko wokungahambelani kweqondo lokushisa elibizwa ngokuba yi "edge effect"
Ukuguquka kwamanzi
Ugxininiso lwamacandelo empendulo nganye akufuneki lutshintshe ngexesha lokusabela ngenxa yokumka komphunga. Ngaphandle koko, kunokwenzeka ukuba kuncinci kakhuluImveliso yePCRinokuveliswa, okanye kungabikho konke konke. Kubalulekile ke ngoko ukucutha amanzi amdaka ngokuqinisekisa itywina elikhuselekileyo. Kule meko, isiciko esivuthayo se-thermocycler kunye nesitywina somkhumbi sisebenza ngesandla. Iinketho ezahlukeneyo zokutywinwa ziyafumanekaIipleyiti zePCR (ikhonkco: Inqaku lokutywina), apho elona tywina lifumaneka ngokutywinwa kobushushu. Okunye ukuvalwa kunokuba kufanelekile, nje ukuba uxinzelelo loqhagamshelwano lwesiciko se-cycler lunokulungiswa kwisitywina esikhethiweyo.
Ukubekwa emgangathweni kwenkqubo kuyasebenza ukuze kugcinwe iziphumo ezichanekileyo nezinokuphinda ziphinde ziphinde ziphinde ziphinde ziphindeke emva kwexesha elide. Oku kubandakanya ukugcinwa rhoqo kwesixhobo ukuqinisekisa ukuba sihlala sikwimeko entle yokusebenza. Zonke izinto ezisetyenziswayo kufuneka zibe zezomgangatho ophezulu ngokuthe gqolo kuwo onke amaqashiso avelisiweyo, kwaye ukufumaneka kwazo okuthembekileyo kufuneka kuqinisekiswe.
Ixesha lokuposa: Nov-29-2022