Nangona imo yoqongqo, imolekyuli enye ye template iza kwanela, iDNA enkulu yeDNA isetyenziselwa i-CRC, umzekelo, ukuya kuthi ga kwi-1 μg ye-1 pg ye-DG ye-DG ye-DNAMID DNA. Imali efanelekileyo ixhomekeke kakhulu kwinani leekopi zokulandelelana kwethagethi, kunye nokuntywila.
Ukuba itemplate encinci kakhulu isetyenziselwa, ukunyuka okufanayo kwinani lemijikelezo yokulungisa iyakufuneka ukufumana inani elaneleyo lemveliso. I-Taq Polymerase esetyenziselwa uninzi lweemvavanyo ze-PCR ze-PCR azichaphazeli umsebenzi wokulungisa (3'-5 'umsebenzi we-elsuclease); Ke, iimpazamo ezenzeka ngexesha lokukhula azinakulungiswa. Ngaphezulu inani lemijikelezo, eyona nto ixhaphakileyo yemveliso efihlakeleyo iya kuba. Ukuba, kwelinye icala, isixa setemplate siphezulu kakhulu, inokwenzeka ye-primers echaza enye (hayi i-artiment enye yeepesenti), kunye nokwakhiwa kwe-daimer ye-primer, iya kukukhokelela kwi-Ampharition ye ziimveliso. Kwiimeko ezininzi, iDNA yodwa kwiinkcubeko okanye kwii-microorganisosm kwaye zaza zasetyenziswa njengeplapri template. Ukucwangciswa kokucocwa, kuyimfuneko ukufumanisa ukubaluleka kwe-DNA ukuba ikwazi ukuchaza ivolumu efunekayo kwi-PCR SETUP. Ngelixa i-agarose gel Electrophoressis inokusebenza ukubonelela ngoqikelelo, le ndlela ilele ekuchanekeni. I-UV-Vis SpectraPhotometry isekwa njengomgangatho wegolide wokuxabana kwee-acid ze-nucleic; Le ngqo ngqo kwaye ke ilula ngokulula kwaye ikhawulezileyo yesampulu ye-260 NM, kwaye uxinzelelo lubalwa ngoncedo lwento yokuguqula.
Ukuba uxinzelelo lwe-DNA lusezantsi kakhulu, nangona kunjalo (<1 μg / ml DSDNA), okanye ukuba ingcolisiwe yimigangatho engama-260 NM (umzekelo, iproteyini), le ndlela iya kufikelela ukusikelwa umda. Kwimeko yoxinzelelo olukhulu kakhulu, ukufundisisa kungekudala kuya kuba singachanekanga ukuba isetyenziswe, kwaye i-sumampution iya kukhokelela kwi (ngamanye amaxesha enkulu) i-overenimetion yexabiso elichanekileyo. Kule meko, ukuxabana kusetyenziswa i-fluorescence kunokwenza enye indlela. Obu buchule busekwe ekusebenziseni i-DEORESCT DYE ebophelela kwi-DSDNA kuphela i-DSDN kuphela i-dacleid ye-nucleic acid kunye nedayi yochulumancinane. Apha, ubukhulu bomqondiso we-fluorescent buhambelana nexabiso le-DNA, kunye nokufumanisa uxinzelelo luvavanywe ngokunxulumene nejika eliqhelekileyo. Izibonelelo zale ndlela ziphumle kubuchwephesha beBond, ebandakanyi iimpembelelo zangaphandle ezingeniswe ngongcoliseko, kunye nokukwazi ukufumana i-DNA ephantsi kakhulu yeDNA. Ukufaneleka kwendlela nayiphi na indlela kuxhomekeke ikakhulu kugxininiso lwesampulu kunye nobunyulu; Kwiimeko ezininzi kusenokucetyiswa ukuba basebenzise zombini iindlela ngokuhambelana.
IXESHA LOKUQALA: UNV-30 ukuya kwi-2022