Sungula isilingo se-PCR SOP ukuze ulinganise ukuziphatha kwabasebenzi bokuhlola.
Umhloli uthobela izinqubo zokusebenza ngokuqinile, futhi unciphisa ukungcoliswa kwe-PCR okungenzeka kubangelwe izici zomuntu noma okuvimbela ukwenzeka kokungcola.Ngaphezu kwalokho, ohlolayo kufanele abe nolwazi oluhambisanayo lobungcweti namakhono, okuhlanganisa ubungoti bemishini ehlobene nokusebenza, ukucacisa yonke inqubo yomsebenzi, ukuphatha izindlela zokwelapha zokungcoliswa nezindlela zokulawula ikhwalithi zaselabhorethri, kanye nokukwazi ukuchaza kahle imiphumela yokuhlolwa.
Sungula ilabhorethri ye-PCR ejwayelekile.
Ilabhorethri ye-PCR ihlukaniswe yaba izindawo ezine ngokomgomo, okuyindawo yokulungisa i-reagent, indawo yokucubungula isampula, indawo yokukhulisa, kanye nendawo yokuhlaziywa komkhiqizo.Izindawo ezimbili zokuqala yizindawo zokwandiswa kwangaphambili, kanti izindawo ezimbili zokugcina yizindawo zokwandiswa.Indawo yokukhulisa i-pre-amplification kanye ne-post-amplification zone kufanele ihlukaniswe ngokuqinile.Izinto zokuhlola, ama-reagents, iphepha lokuqopha, amapeni, izinto zokuhlanza, njll., zingageleza kuphela zisuka endaweni yokukhulisa isandiso ziye endaweni yangemuva kokukhulisa, okungukuthi, zisuka endaweni yokulungiswa kwe-reagent → indawo yokucubungula isampula → indawo yokukhulisa → indawo yokuhlaziya umkhiqizo wokukhulisa, futhi akumelwe agezele emuva .Ukugeleza komoya elabhorethri kufanele futhi kugeleze ukusuka endaweni yokukhulisa isandiso kuya endaweni yangemuva kokukhulisa, futhi ungagelezi uhlehle.Idizayini efanele yelabhorethri ye-PCR ikhonjiswe ngezansi:
Umfanekiso A: Imodi yokusetha elabhorethri ye-PCR ekahle enengcindezi engalungile egumbini le-buffer
Umfanekiso B: Imodi yokusetha ilabhorethri ye-PCR ekahle enokucindezela okuhle egumbini le-buffer
Imidwebo yokusetha ilabhorethri ye-PCR enikezwe kuMdwebo A kanye noFigure B kufanele ibe imodi yokusetha ekahle kakhudlwana, futhi ilabhorethri enemibandela ingabhekisela kule modi ukuze iklanywe.Kumalabhorethri ajwayelekile, kunconywa ukuthi indawo yokukhulisa i-PCR kanye nendawo yokuhlaziya umkhiqizo ingahlukaniswa, futhi ukuvulwa kwekhava kufanele kuncishiswe ngangokunokwenzeka endaweni yokulungiselela isampula kanye nendawo yokukhulisa i-PCR.Khumbula: Imikhiqizo kanye nezinto ezisetshenziswayo zokuhlola endaweni yokuhlaziya umkhiqizo zinqatshelwe ngokuphelele ukuthi ziyiswe endaweni yokulungiselela isampula kanye nendawo yokukhulisa i-PCR.
Uma ilabhorethri ihlola kuphela i-PCR nokuhlonza, kuyanconywa ukuthi kusetshenziswe i-fluorescent quantitative PCR esikhundleni se-PCR evamile.
Imiphumela yokutholwa kwe-PCR yobuningi be-Fluorescence ingaqoqwa futhi ihlaziywe ngamasignali e-fluorescence, ngakho-ke asikho isidingo sokuvula isivalo se-electrophoresis ngemva kokusabela, esigwema ukungcoliswa komkhiqizo we-PCR okubangelwa ukuvuza kwemikhiqizo yokusabela ukuze kwakhe ama-aerosol.Uma ukhuphula inani lokuvuleka kwe-cap ngesikhathi sesinyathelo sokulayisha i-gel electrophoresis, ukungcoliswa kwe-aerosol kungenzeka.Kunconywa ukukhuthaza ukusetshenziswa kwe-PCR yomthamo futhi kancane kancane kuthathelwe indawo i-PCR yekhwalithi.
Isistimu yokungcoliswa komkhiqizo we-UNG elwa ne-PCR isetshenziselwa ukusabela kwe-PCR.
Uhlelo lusebenzisa i-dUTP esikhundleni se-dTTP.Ngemva kokusabela kwe-PCR, yonke imikhiqizo ye-PCR (izingcezu ze-DNA) ihlanganiswa ne-dUTP;emzuliswaneni olandelayo wokusabela kwe-PCR, i-enzyme ye-UNG engezwe kusistimu ifakwa ku-37°C imizuzu emi-5 ngaphambi kwe-PCR, engakwazi ukwehlisa isithunzi zonke izingcezu ze-DNA eziqukethe i-dUTP, bese yenze ukusabela kwe-PCR.Lokhu kungasusa ngokuphelele ukungcoliswa kwe-aerosol okubangelwa imikhiqizo ye-PCR.Umphumela uboniswa emfanekisweni ongezansi:
Qaphela: Ochungechungeni lwe-PCR oluqondile, ungakhetha uchungechunge lwemikhiqizo yohlelo lokungcoliswa komkhiqizo olulwa ne-PCR lwe-Foregene.Phakamisa
Ezilabhorathri ezihlola i-genotyping enkulu, kunconyelwa kakhulu ukusebenzisa uhlelo lokungcolisa umkhiqizo we-UNG anti-PCR ukuhlola izinto ezisebenza ngokusebenza kabusha ngokungeziwe ekwakhiweni kwamalabhorethri anengqondo.
Isikhumbuzi: Ukusetshenziswa kwalolu hlelo akukwazi ukususa ukungcoliswa komkhiqizo we-PCR osekudaliwe.Ngakho-ke, uhlelo lwe-UNG kufanele lusetshenziswe ekuqaleni kokuhlolwa okufanele, futhi uhlelo lwe-UNG kufanele lusetshenziselwe ukukhulisa i-PCR, ukuze kuvinjelwe ukungcoliswa kwemikhiqizo ye-PCR I-false positive.
Kunconywa ukusebenzisa uhlelo lwe-Direct PCR-UNG lwe-Foregene lapho kwenziwa ukuhlola okukhulu, okufana nalokhu:
I-Plant Leaf Direct PCR Kit-UNG;
Plant Seed Direct PCR Kit-UNG;
I-Animal Tissue Direct PCR Kit-UNG;
I-Mouse Tail Direct PCR Kit-UNG;
I-Zebra Fish Direct PCR Kit-UNG.
Lolu chungechunge lwamakhithi avela ku-Foregeneayikwazi nje ukwenza ukutholwa kwe-PCR ngokushesha nangesilinganiso esikhulu, kodwa futhi ivimbela ngempumelelo futhi ilawule ukungcoliswa komkhiqizo we-PCR.
Isikhathi sokuthumela: Mar-19-2021
