1:Shintsha izinto zokuhlola ngesikhathi

Setha (NTC) isilawuli esinegethivu bese usiphinda izikhathi eziningi.Uma sekutholakele ukuthi kukhona ukungcoliswa komkhiqizo we-PCR elabhorethri, buyisela zonke izinto zokuhlola ngesikhathi.Okufana nokuthi: hlambulula kabusha futhi ulungise ama-primers, phinda ugcwalise ithiphu le-pipette, ishubhu le-EP, i-ddH2O, njll., buyisela i-pipette entsha, bese uboleka okwesikhashana ezinye izindawo zokucwaninga ukuze zenze izivivinyo ze-PCR.Ilabhorethri engcolile izongena umoya futhi ifakwe imisebe ye-ultraviolet kuze kube yilapho ukungcoliswa komkhiqizo we-PCR kuqedwa ngaphambi kokuqhubeka nokuhlola.

2: Nweba isikhathi sokuchayeka kwe-UV

Kuyaqapheleka ukuthi ukususa ukungcola kwe-DNA, imisebe ye-ultraviolet evamile kufanele inwetshwe ngamahora ama-2 kunokujwayelekile.Noma kunjalo, umthelela wokukhanya kwe-UV ekususeni izingcezu ezincane (ngaphansi kuka-200bp) zokungcola kwe-DNA awukabi muhle.

I-Ultraviolet wavelength (nm) ivamise ukuba ngu-254/300nm, futhi yanele ukukhanyisela imizuzu engama-30.Kufanele kuqashelwe ukuthi lapho ukhetha i-UV ukuze kuqedwe ukungcoliswa kwemikhiqizo esele ye-PCR, ubude bomkhiqizo we-PCR kanye nokusatshalaliswa kwezisekelo ngokulandelana komkhiqizo kufanele kucatshangelwe.Ukukhanya kwe-UV kusebenza kuphela ezincezuni ezinde ezingaphezu kuka-500 bp, futhi kunomphumela omncane kuzingcezu ezimfushane.

Ngesikhathi sokukhanya kwe-UV, izisekelo ze-pyrimidine kumkhiqizo we-PCR zizokwakha ama-dimers.Lawa ma-dimers anganqamula isandiso, kodwa akuwona wonke ama-pyrimidines ochungechungeni lwe-DNA angakha ama-dimers, futhi ukukhanya kwe-UV kungaphinda kuphule ama-dimers..Izinga lokwakheka kwe-dimer lincike kubude beza be-UV, uhlobo lwe-pyrimidine dimer kanye nokulandelana kwama-nucleotide ancikene nesayithi le-dimer.Ngakho-ke, uma izingcezwana ezithuthukisiwe ze-PCR zizincane, kutuswa ukusebenzisa uhlelo lokungcolisa umkhiqizo lwe-UNG anti-PCR.

3:Izikhukhula zokungcoliswa kwe-DNA ezisetshenziswa kakhulu

Ama-aerosols akhiqizwa kalula lapho ama-pipette engeziwe, okunzima ukuwagwema, futhi azoxazulula ngokushesha.Ngakho-ke, akungabazeki ukuthi kuyisinqumo esihle ukusebenzisa kaningi izinto ezikhethekile zokungcolisa i-DNA ukuvimbela ukusabalala kokungcola kwe-DNA.

4:Sebenzisa uhlelo lwe-UNG lokulwa nokunukubezeka

Ngemva kokususwa kokungcola komkhiqizo we-PCR, ilabhorethri yokuhlola ingasebenzisa isistimu yokungcolisa umkhiqizo we-UNG ye-PCR ukuvimbela ukungcoliswa komkhiqizo we-PCR.Ngokujwayelekile amalabhorethri, ungenza izingxenye zokuhlola ezilula, uhlukanise ngokuqinile indawo yomkhiqizo we-PCR kwezinye izindawo, umise imithetho nemithetho ethile yaselabhorethri, futhi wenze ukuqeqeshwa okufanelekile ukuvimbela ukwenzeka kokungcoliswa komkhiqizo we-PCR.

Izincomo: Ukusungula ilabhorethri ye-PCR enengqondo, ukugcina indawo enhle ye-PCR, ukwenza izinqubo ezijwayelekile zokusebenza kwe-PCR, kanye nokuhlakulela ukuqwashisa okuqinile kokusebenza kwabahloli kuyizihluthulelo zokuvimbela noma zokunciphisa ukungcoliswa kokuhlolwa kwe-PCR.