Animal Tissue Direct PCR kit (popanda Sampling Tools) Protocol
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 kuchotsedwa kwa mapuloteni ndi RNA zomwe zimafunikira, ndipo DNA yotulutsidwa ingagwiritsidwe ntchito ngati template ya PCR reaction.
2 × PCR YosavutaTMMix ili ndi kulolerana kwamphamvu kwa PCR reaction inhibitors, ndipo imatha kugwiritsa ntchito lysate yachitsanzo kuti iyesedwe ngati template yokulitsa bwino komanso yeniyeni.Reagent iyi ili ndi Foregene D-Taq DNAPolymerase, dNTPs, MgCl2, zotchingira zochita, PCR optimizer ndi stabilizer.
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.
Zofotokozera
50×20µl rxns, 200×20µl rxns, 500×20µl rxns, 2000×20µl rxns
Zida zamagulu
Gawo I | Bafa AL |
Foregene Protease | |
6 × DNA Loading Buffer | |
Gawo II | 2 × PCR YosavutaTMSakanizani |
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.
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 EasyTMSakanizani akhoza kusungidwa kwa miyezi 12 pa -20℃;ngati imagwiritsidwa ntchito pafupipafupi, imathanso kusungidwa pa 4℃kusungirako kwakanthawi kochepa (kugwiritsa ntchito mkati mwa masiku 10).