Ngwa anụ ahụ Direct PCR kit-UNG (na-enweghị ihe nlele) Protocol
Nkọwa
Ngwa a na-eji sistemụ nchekwa lysis pụrụ iche iji wepụta DNA genomic ngwa ngwa site na nlele anụ ahụ anụ ahụ maka mmeghachi omume PCR, yabụ ọ dabara adaba maka nnwale mkpụrụ ndụ ihe nke ukwuu.
Usoro nke ịtọhapụ DNA genomic site na nchekwa lysis na-agwụ n'ime nkeji 10-30 na 65 Celsius.Ọ nweghị usoro ndị ọzọ dị ka protein na mwepụ RNA achọrọ, yana DNA achọpụtara ewepụtara enwere ike iji dị ka ndebiri maka mmeghachi omume PCR.
2 × PCR dị mfeTMMix (UNG) na-eji dUTP kama dTTP na ndabere nke 2 × PCR EasyTMGwakọta, ma gbakwunye UNG enzyme (Uracil-N-glycosylase) nke nwere ike imebi template nwere dUTP n'otu oge.Tupu mmeghachi omume PCR, a na-eji enzyme UNG mebie ngwaahịa PCR nwere uracil.Enzyme UNG agaghị enwe mmetụta ọ bụla na ndebiri nke na-enweghị uracil, si otú a na-ahụ maka nkọwapụta na izi ezi nke mmụba na igbochi ohere nke mmetọ ngwaahịa PCR n'oge ule mkpụrụ ndụ ihe nketa buru ibu.
D-Taq DNA polymerase bụ DNA polymerase nke Foregene mepụtara maka mmeghachi omume PCR kpọmkwem.D-Taq DNA polymerase nwere nnabata siri ike maka ọtụtụ ihe mgbochi mmeghachi omume PCR, ma nwee ike ịbawanye ọnụọgụ DNA nke ọma na sistemụ mmeghachi omume dị mgbagwoju anya, yana ọsọ nkwalite nwere ike iru 2Kb / min, nke dabara adaba maka mmeghachi omume PCR Direct.
Ngwa ngwa
Akụkụ I | Ihe nchekwa AL |
Protease nke mbụ | |
Ihe nchekwa 6 × DNA na-ebufe | |
Akụkụ II | 2 × PCR dị mfeTMNgwakọta (UNG) |
Atụmatụ & uru
■Ọ dịghị ọcha DNA na-ewe oge na ọnụ
∎ Ihe nlele achọrọ dị ntakịrị, ebe ọ bụ na anụ ahụ 5mg nwere ike ịnwale.
∎ Ọ dịghị ọgwụgwọ pụrụ iche dị ka egweri na ịzọpịa a chọrọ, ma ọrụ ahụ dị mfe.
∎ Sistemu PCR kachasị mma na-eme ka PCR nwee nkọwa dị elu yana nnabata siri ike nye ndị na-egbochi mmeghachi omume PCR.
■ Sistemụ PCR na-egbochi mmetọ 2×PCR dị mfeTMNgwakọta (UNG) na-ewepụ mmetọ nke ngwaahịa PCR nke ọma wee hụ na nkọwapụta na izi ezi nke nkwalite.
Ngwa ngwa
Ngwa: ụdị anụ ahụ dị iche iche.
DNA ewepụtara na lysis sample: naanị ejiri ya dị ka ndebiri PCR.
Enwere ike iji ngwa ahụ maka ebumnuche ndị a: njirimara transgenosis, genotyping anụmanụ, wdg.
Usoro ọrụ
Nchekwa na Shelf ndụ
Ekwesịrị ịchekwa akụkụ I nke ngwa a na 2-8℃.
Enwere ike ịchekwa reagent Buffer AL maka ọnwa 12 n'okpuru ọnọdụ akọrọ;Enwere ike ịchekwa ya na 2-8℃maka ogologo nchekwa.
Reagent Foregene Protease nwere usoro pụrụ iche.Iji hụ na ọrụ ya na nkwụsi ike ya, biko chekwaa ya na 4℃maka ọnwa iri na abụọ.
Reagent 6×Enwere ike ịchekwa ihe nchekwa DNA na 4℃ma ọ bụ -20℃ogologo oge.
Ekwesịrị ịchekwa akụkụ II nke ngwa a na -20℃.
Reagent 2×PCR dị mfeTMNgwakọta (UNG), ọ bụrụ na a na-ejikarị ya eme ihe, enwere ike ịchekwa ya na 4℃maka nchekwa obere oge (jiri elu n'ime ụbọchị 10).