Kodi tikuyenera kuwonjezera bwanji PCR yanga?

Ngakhale mu lingaliro, mamolekyu amodzi a template amakhala okwanira, ambiri ochulukirapo amagwiritsidwa ntchito ngati 1 μg ya genomical DNA ndi imodzi ya Plasmid DNA. Kuchuluka koyenera kumadalira kwambiri kuchuluka kwa makope a zomwe akufuna kutsatira, komanso kuvuta kwake.

Ngati template yaying'ono kwambiri imagwiritsidwa ntchito, kuwonjezeka kofanana munthawi ya ma exmation adzafunika kuti mupeze mankhwala okwanira. A TAQ Polymease yomwe imagwiritsidwa ntchito poyesa kwambiri PCR siyikuwonetsa ntchito yowongolera (3 '-5' exukolo Chifukwa chake, zolakwika zomwe zimachitika pa Kukula sizingakonzedwe. Kuchuluka kwazomwe zimachitika, zomwe zimafalikira kwambiri. Ngati, kumbali inayo, kuchuluka kwa template kumakhala kokwera kwambiri, kuthekera kwa oyambira ena (palibe zana limodzi), komanso kuchuluka kwa primer, komwe kumabweretsa, komwe kumabweretsa kuphatikizira kwa zopangidwa ndi. Nthawi zambiri, DNA imapatula zikhalidwe zam'manja ndipo pambuyo pake zimagwiritsidwa ntchito ngati template ya pcr. Kutsatira, ndikofunikira kudziwa kuchuluka kwa DNA kuti athe kufotokozera voliyumu yomwe imafunikira kuti PCR Setep. Ngakhale Agarose Gel Electrophoresis imatha kupereka kuti iperekenso kuyerekezera, njirayi siyolondola. UV-vis Sportrophirotometry yakhazikitsidwa ngati muyezo wagolide wa kuchuluka kwa ma acid a nuclec; Chifukwa chake molunjika komanso mosavuta komanso njira yofulumira imayesa kuyamwa kwa zitsanzo za pa 260 nm, ndipo ndende imawerengeredwa mothandizidwa ndi chinthu chosintha.

Ngati DNA Yokhazikika imakhala yotsika kwambiri, komabe (<1 <) Pankhani yotsika kwambiri, kuwerenga posachedwa kungakhale kolakwika kwambiri kuti igwiritsidwe ntchito, ndipo kuipitsidwa kumadzetsa (nthawi zina kumabweretsanso) kuchuluka kwa mtengo wake. Pankhaniyi, kuchuluka pogwiritsa ntchito anzeru kumatha kubweretsanso njira ina. Njirayi imakhazikika pakugwiritsa ntchito utoto wa fluoressesy womwe umamangiriza ku DSDNA yokhayo yomwe imapangitsa chidwi cha nuclena ophatikizika ndi kuwala, ndipo kenako imawunikira pang'ono pang'ono. Apa, kukula kwa chizindikiro cha fluorescent ndikofanana ndi kuchuluka kwa DNA, ndipo pofuna kudziwa kuchuluka kwazomwe zimawunikidwa mogwirizana ndi kupindika. Ubwino wa njirayi umapuma pankhani ya chomangirachi, chomwe sichimasiyidwa zakunja zomwe zimayambitsidwa ndi kuipitsidwa, komanso pazomwe zimayambitsa kuzindikira zotsika kwambiri za DNA. Kuyenerera kwa njira iliyonse kumadalira makamaka pa zitsanzo za kuvutika ndi chiyero; Nthawi zambiri zitha kukhala bwino kugwiritsa ntchito njira zonsezi.