ʻO Uracil DNA Glycosylase
ʻO Uracil-DNA Glycosylase (UNG a i ʻole UDG) he clone recombinant o E.coli me ke kaumaha molekala o 25 kDa.Hoʻopiʻi ia i ka hoʻokuʻu ʻana o ka uracil manuahi mai ka uracil i loaʻa i ka DNA stranded hoʻokahi a me ke kaula pālua, a ʻaʻohe hana kūʻē i ka RNA, a hiki ke hoʻohana ʻia e pale i ka haumia o nā huahana hoʻonui PCR.Hoʻokumu ʻia ke kumu o ka hana ma ka ʻoiaʻiʻo inā ua hoʻololi ʻia ka dUTP no ka dTTP i ka hopena PCR a ua hoʻokumu ʻia kahi huahana PCR amplification i loaʻa nā kumu dU, hiki i ka enzyme koho ke uhaki i ka paʻa glycosidic o nā kumu U i hoʻokahi-stranded a pālua-stranded. DNA a hoʻohaʻahaʻa i ka huahana hoʻonui PCR.
Manaʻo Noi
Hoʻonui Kāohi ʻana i ka hoʻohaumia
Kūlana Waihona
-20°C no ka mālama ʻana i ka wā lōʻihi, pono e hui pū ʻia ma mua o ka hoʻohana ʻana, e pale i ka hoʻoheheʻe pinepine ʻana.
Paʻa mālama
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Wehewehe Unite
ʻO ka nui o ka enzyme e pono ai e hoʻohaʻahaʻa i 1µg o ka DNA kaula hoʻokahi i loaʻa nā kumu dU i 1 hola ma 37°C he 1 ʻāpana.
Ka Mana Mana
1.ʻOi aku ka maʻemaʻe electrophoretic SDS-PAGE ma mua o 98%
2.Ka hoʻonui ʻana i ka naʻau, ka hoʻomalu ʻana i ka pūʻulu, kūpaʻa
3.Ma hope o ka mālama ʻia ʻana o 1U o UNG ma 50 ℃ no 2 mau minuke, pono e hoʻohaʻahaʻa loa ʻia ka template i loaʻa iā U ma lalo o 103 kope a ʻaʻole hiki ke hana ʻia kahi huahana amplification.
4.ʻAʻohe hana nuclease exogenous
Nā kuhikuhi
| Nā ʻāpana | Volume (μL) | Hoʻoholo hope |
| 10 × PCR Buffer (dNTP manuahi, Mg²+manuahi) | 5 | 1× |
| nā dUTP (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (hoʻololi i ka dTTP) | - | 200-600 μM |
| 25 mM MgCl2 | 2-8 μL | 1-4 mM |
| 5 U/μL Taq | 0.25 | 1.25 U |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 25 × Hui Pūmuaa | 2 | 1× |
| Papahana | - | ＜1μg/pane |
| ddH₂O | I ka 50 | - |
Nānā: a: Inā hoʻohana ʻia no qPCR/qRT-PCR, pono e hoʻohui ʻia ka ʻimi fluorescent i loko o ka ʻōnaehana pane.ʻO ka maʻamau, hiki ke hāʻawi i nā hopena maikaʻi i kahi ʻano kumu mua o 0.2 μM;i ka wā ʻilihune ka hana ʻana, hiki ke hoʻoponopono ʻia ke kumu kumu ma ka laulā o 0.2-1 μM.ʻO ka maʻamau, ʻoi aku ka maikaʻi o ka ʻimi noiʻi ma ka laulā o 0.1-0.3 μM.Hiki ke hana ʻia nā hoʻokolohua gradient concentration no ka ʻimi ʻana i ka hui maikaʻi loa o ka primer a me ka probe.
Nā memo
1.Hiki ke hoʻohana ʻia ka enzyme UNG e wehe i nā huahana amplification dUTP i hoʻohaumia ʻia mai ka ʻōnaehana hopena ma mua o ka hopena o ka PCR amplification, a laila e pale aku i nā hopena maikaʻi ʻole ma muli o ka haumia o ka huahana.
2.ʻO ka mahana maikaʻi loa no ka enzyme UNG e hoʻohana ʻia i ka hopena PCR anti-contamination ma ke ʻano he 50 ℃ no 2 mau minuke;ʻO ke kūlana inactivation he 95 ℃ no 5mins.
3.E hōʻalo i ka hoʻoheheʻe pinepine ʻana, a mai hōʻike i nā loli wela nui.
4.ʻO nā ʻano genes e hoʻonui ʻia he ʻokoʻa ka hoʻohana pono ʻana o ka dUTP a me ka naʻau i ka enzyme UNG, no laila, inā ʻo ka hoʻohana ʻana i ka ʻōnaehana UNG e alakaʻi i ka emi ʻana o ka ʻike ʻike, pono e hoʻoponopono ʻia ka ʻōnaehana hopena, inā makemake ʻoe i ke kākoʻo ʻenehana, e ʻoluʻolu e kelepona. ko makou hui.
