Animal Tissue Direct PCR kit-UNG (popanda Sampling Tools) Protocol
Kufotokozera Kwa Zida:
Gwiritsani ntchito mwachindunji minofu ya nyama ya lysate ngati template yakukulitsa kwa PCR, popanda kutulutsa kosiyana kwa DNA, kufulumira komanso kukhudzika kwambiri.Adawonjezera UNG anti-pollution system kuti athetse zolakwika.
◮Palibe kuyeretsa kwa DNA kotenga nthawi komanso kokwera mtengo komwe kumafunikira.
◮Zofuna zachitsanzo ndizochepa, ingotenga mbeu imodzi.
◮Palibe mankhwala apadera monga kugaya ndi kuphwanya omwe amafunikira, ndipo ntchitoyo ndi yosavuta.
◮Makina okhathamiritsa a PCR amathandizira PCR kukhala ndi mawonekedwe apamwamba komanso kulolerana mwamphamvu kwa PCR reaction inhibitors.
Kufotokozera
Chidachi chimagwiritsa ntchito njira yapadera ya lysis buffer kuti itulutse mwachangu ma DNA amtundu wa nyama pazochitika za PCR, kotero ndiyoyenera kuyesa kwakukulu kwa majini.
Njira yotulutsa DNA ya genomic kuchokera ku lysis buffer imatsirizidwa mkati mwa mphindi 10-30 pa 65 ° C.Palibe njira zina monga puloteni ndi kuchotsa RNA zomwe zimafunikira, ndipo DNA yotulutsidwa ingagwiritsidwe ntchito ngati template ya PCR reaction.
2 × PCR YosavutaTMMix (UNG) amagwiritsa ntchito dUTP m'malo mwa dTTP pamaziko a 2× PCR EasyTMSakanizani, ndikuwonjezera UNG enzyme (Uracil-N-glycosylase) yomwe ingawononge template yomwe ili ndi dUTP nthawi yomweyo.PCR isanachitike, puloteni ya UNG imagwiritsidwa ntchito kusokoneza mankhwala a PCR okhala ndi uracil.Enzyme ya UNG sidzakhala ndi zotsatira pa template yomwe ilibe uracil, potero imatsimikizira kutsimikizika ndi kulondola kwa kukulitsa ndikuletsa kuthekera kwa kuipitsidwa kwa zinthu za PCR pakuyesa kwakukulu kwa majini.
D-Taq DNA polymerase ndi DNA polymerase yopangidwa mwapadera ndi Foregene kuti achite mwachindunji PCR.D-Taq DNA polymerase ili ndi kulolerana kwamphamvu kwa mitundu yosiyanasiyana ya PCR reaction inhibitors, ndipo imatha kukulitsa bwino kuchuluka kwa DNA m'machitidwe osiyanasiyana ovuta kuchita, ndipo liwiro lokulitsa limatha kufikira 2Kb/mphindi, lomwe ndiloyenera kwambiri kuchitapo kanthu kwa Direct PCR.
Zida zamagulu
| Gawo I | Bafa AL |
| Foregene Protease | |
| 6 × DNA Loading Buffer | |
| Gawo II | 2 × PCR YosavutaTMMix(UNG) |
Mbali & ubwino
■Palibe nthawi yambiri komanso yokwera mtengo yoyeretsa DNA
■ Kufuna kwachitsanzo ndikochepa, monga momwe minofu yanyama ya 5mg ingayesedwe.
■ Palibe chithandizo chapadera monga kugaya ndi kuphwanya chofunika, ndipo opaleshoniyo ndi yosavuta.
■ Makina a PCR okometsedwa amapangitsa PCR kukhala ndi mawonekedwe apamwamba komanso kulolerana mwamphamvu kwa PCR reaction inhibitors.
■ Pulogalamu ya PCR yolimbana ndi kuipitsidwa 2×PCR EasyTMMix (UNG) imathetsa bwino kuipitsidwa kwa zinthu za PCR ndikuwonetsetsa kutsimikizika ndi kulondola kwa kukulitsa.
Zosintha za Kit
Ntchito: zosiyanasiyana nyama zimakhala.
DNA yotulutsidwa kuchokera ku lysis yachitsanzo: imagwiritsidwa ntchito ngati template ya PCR.
Zidazi zitha kugwiritsidwa ntchito pazifukwa izi: kuzindikira transgenosis, genotyping ya nyama, ndi zina.
Kuyenda kwa ntchito
Kusungirako ndi moyo wa alumali
Gawo I la zida izi ziyenera kusungidwa pa 2-8℃.
The reagent Buffer AL ikhoza kusungidwa kwa miyezi 12 pansi pauma;ikhoza kusungidwa pa 2-8℃posungirako nthawi yayitali.
Reagent Foregene Protease ili ndi njira yapadera.Kuti muwonetsetse kuti ikugwira ntchito komanso kukhazikika, chonde sungani pa 4℃kwa miyezi 12.
Reagent 6×DNA Loading Buffer ikhoza kusungidwa pa 4℃kapena -20℃kwa nthawi yayitali.
Gawo II la zida izi ziyenera kusungidwa pa -20℃.
Reagent 2×PCR EasyTMMix(UNG), ngati imagwiritsidwa ntchito pafupipafupi, imathanso kusungidwa pa 4℃kusungirako kwakanthawi kochepa (kugwiritsa ntchito mkati mwa masiku 10).
