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1:Buyisela izibonelelo zovavanyo ngexesha

iindaba812 (1) 

Cwangcisa (NTC) ulawulo olungalunganga kwaye uluphinde amaxesha amaninzi.Nje ukuba kufunyaniswe ukuba kukho ungcoliseko lwemveliso yePCR elabhoratri, buyisela zonke izinto zovavanyo ngexesha.Okufana noku: phinda uhlambulule kwaye ulungiselele iiprimers, phinda ucofe incam yepipette, ityhubhu ye-EP, i-ddH2O, njl., buyisela i-pipette entsha, kwaye uboleke okwethutyana ezinye iilabhoratri ukwenza imifuniselo ye-PCR.Ilabhoratri engcolisekileyo iya kufakwa umoya kwaye ifakwe imisebe ye-ultraviolet de ungcoliseko lwemveliso yePCR lupheliswe phambi kokuba kuqhutyekwe novavanyo.

2: Yandisa ixesha lokuvezwa kwe-UV

iindaba812 (2)

Kuyafaneleka ukuba uqaphele ukuba ukususa ukungcola kwe-DNA, imisebe ye-ultraviolet rhoqo kufuneka yandiswe ngeeyure ezingama-2 kunesiqhelo.Nangona kunjalo, umphumo we-UV irradiation ekususeni amaqhekeza amancinci (ngaphantsi kwe-200bp) yokungcola kwe-DNA ayikalungile.

I-Ultraviolet wavelength (nm) ngokuqhelekileyo yi-254/300nm, kwaye yanele ukukhupha i-30 imizuzu.Kufuneka kuqatshelwe ukuba xa ukhetha i-UV ukuphelisa ukungcoliswa kweemveliso ze-PCR ezishiyekileyo, ubude bemveliso ye-PCR kunye nokusabalalisa iziseko ngokulandelelana kwemveliso kufuneka kuthathelwe ingqalelo.Ukukhanya kwe-UV kusebenza kuphela kumaqhekeza amade ngaphezulu kwe-500 bp, kwaye kunempembelelo encinci kumaqhekeza amafutshane.

Ngexesha lokukhanya kwe-UV, iziseko ze-pyrimidine kwimveliso ye-PCR ziya kwenza i-dimers.Ezi dimers zinokuphelisa ukwandiswa, kodwa ayizizo zonke iipyrimidines kwikhonkco le-DNA ezinokwenza i-dimers, kunye ne-UV irradiation inokwaphula i-dimers..Iqondo lokubunjwa kwe-dimer lixhomekeke kwi-wavelength ye-UV, uhlobo lwe-pyrimidine dimer kunye nokulandelelana kwee-nucleotides ezikufutshane ne-dimer site.Ngoko ke, ukuba i-PCR i-amplified fragments incinci, kuyacetyiswa ukuba usebenzise inkqubo ye-UNG ye-anti-PCR yokungcoliseka kwemveliso.

3:Ezixhaphakileyo zisetyenziswa kwiDNA yokungcoliseka kwendalo

iindaba812 (3)

I-aerosols iveliswa ngokulula xa i-pipettes yongezwa, okunzima ukuyiphepha, kwaye iya kuhlala ngokukhawuleza.Ngoko ke, ngokungathandabuzekiyo lukhetho olulungileyo lokusebenzisa rhoqo i-DNA ekhethekileyo yongcoliseko lwe-scavengers ukuthintela ukusasazeka kongcoliseko lwe-DNA.

4: Sebenzisa inkqubo ye-UNG yokuchasana nongcoliseko

iindaba812 (4)

Emva kokuba ungcoliseko lwemveliso ye-PCR lususiwe, ilabhoratri yovavanyo inokusebenzisa inkqubo ye-UNG yokuchasana ne-PCR yokungcoliseka kwemveliso ukuthintela ukungcoliseka kwemveliso ye-PCR.Kwiilabhoratri ngokubanzi, unokwenza izahlulo ezilula zokulinga, ukwahlula ngokungqongqo indawo yemveliso yePCR kwezinye iindawo, ukuseka imithetho nemimiselo ethile zelebhu, kwaye uqhube uqeqesho olufanelekileyo ukunqanda ukwenzeka kokungcoliseka kwemveliso yePCR.

Izindululo: Ukuseka ilabhoratri ye-PCR enengqiqo, ukugcina imeko-bume ye-PCR elungileyo, ukuqulunqa iinkqubo zokusebenza ezisemgangathweni ze-PCR, kunye nokukhulisa ulwazi olunzulu lokusebenza kwabavavanyi zizitshixo zokuthintela okanye zokunciphisa ungcoliseko lwemifuniselo ye-PCR.


Ixesha lokuposa: Aug-12-2021