Te Pawera UNG
Ko te UNG (TS-UNG) Te Maamaa he mea whiwhi ma te whakahuatanga whakahiato i roto i te E. coli.Ka whakakorikori te whākōkī i te tukunga o te uracil kore utu mai i te uracil-kei roto i te DNA kotahi me te whenu-rua, ka kore e kaha ki te RNA.Ka whakatauritea ki te UNG enzyme o E. coli ira takenga mai, he nui ake te mahi a TS-UNG enzyme i te iti o te mahana (20 ℃~37 ℃) me te werawera me te ngawari ki te inactivated (50 ℃), ka karo i te paheketanga o te whakakaha dUTP-kei roto. nga hua i te pāmahana rūma na te toenga mahi ka noho pea i muri i te korenga o te UNG enzyme tikanga.Na reira, ehara i te mea e pai ana te enzyme TS-UNG mo te tauhohenga aukati PCR contamination, engari he pai ano hoki ki te kaupapa whakakaha RT-PCR ka taea te whakamahi i roto i te tauhohenga aukati parahanga RT-PCR.
Taupānga Manakohia
Ko te Whakanuia te Whakanuia
Tikanga Rokiroki
-20°C mo te rokiroki mo te wa roa, me uru pai i mua i te whakamahi, karohia te whakamao-kore-roa.
Pupuri putunga
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Whakamaramatanga Waeine
Ko te nui o te whākōkī e hiahiatia ana hei whakaheke i te 1µg o te DNA whenu kotahi kei roto nga turanga dU i roto i te 1 haora i te 37°C he 1 waeine mahi (U).
Mana Kounga
1.SDS-PAGE te parakore electrophoretic nui ake i te 98%
2.Ko te mahi whakaheke, te mana o te puranga-ki-te-popu, te pumau
3.Karekau he ngohe karihi o waho, karekau he endonuclease o waho, he poke exonuclease ranei.
Tohutohu
| Waehanga | Rōrahi (μL) | Te kukū whakamutunga |
| 10 × PCR Buffer (dNTP kore utu, Mg²+kore utu) | 5 | 1× |
| DUTP (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (whakakapi i te dTTP) | - | 200-600 μM |
| 25 mM MgCl2 | 2-8 μL | 1-4 mM |
| 5 U/μL Taq | 0.25 | 1.25 U |
| 1 U/μLTS-UNG | 0.5 (0.1-0.5) | 0.5 U (0.1-0.5U) |
| 25 × Whakaranu Tuatahia | 2 | 1× |
| tauira | - | ＜1μg/hohenga |
| ddH₂O | Ki te 50 | - |
Tuhipoka: a: Mēnā ka whakamahia mō te qPCR/qRT-PCR, me tāpiri te rangahau kōaro ki te pūnaha tauhohenga.Ko te tikanga, ka taea e te kuhanga tuatahi o te 0.2 μM te whakaputa hua pai;ina he kino te mahi tauhohenga, ka taea te whakatika te kukū tuatahi i roto i te awhe o te 0.2-1 μM.I te nuinga o te wa, ka arotauhia te kukū rangahau i roto i te awhe o te 0.1-0.3 μM.Ka taea te whakahaere i nga whakamatautau rōnaki ki te kimi i te huinga pai o te tuatahi me te rangahau.
Notes
1.Ko te pāmahana tauhohenga tino pai o TS-UNG whākōkī he āhua iti, a ka taea te papaitia i roto i te whānuitanga o 20 ℃ ~ 37 ℃, te inenga o te whākōkī me te wa tauhohenga e taea te papaitia i roto i te whānuitanga o 0.1 ~ 0.5 U, 5~ 10 min;ā, ka taea te whakakore i te whākōkī i roto i te tukanga o te whakawhiti whakamuri.
2.He pai mo te PCR me te RT-PCR hei aukati i te poke.
3.A ape i te whakamaroke i nga wa maha, kaua hoki e pa ki nga rereketanga o te pāmahana.
4.Ko nga ira rereke hei whakanui ake he rereke te kaha o te whakamahi o te dUTP me te aro ki te UNG enzyme, na, mena ka heke te maaramatanga o te punaha UNG ki te whakaheke i te maarama, me whakatika te punaha tauhohenga me te arotau, mena ka hiahia koe ki te tautoko hangarau, me waea atu. to tatou kamupene.
