Uracil DNA Glycosylase
Ko te Uracil-DNA Glycosylase (UNG, ko te UDG ranei) he kowhatu recombinant o E.coli me te taumaha ngota o te 25 kDa.Ka whakakorikori i te tukunga o te uracil kore utu mai i te uracil-kei roto i nga whenu kotahi me nga whenu-rua te DNA, a ka kore e kaha ki te RNA, ka taea te whakamahi hei aukati i te poke o nga hua whakakaha PCR.Ko te kaupapa o te mahi i runga i te meka mena ka whakakapihia te dUTP mo te dTTP i roto i te tauhohenga PCR ka hangaia he hua whakakaha PCR kei roto nga turanga dU, ka taea e te enzyme te wehe i te here glycosidic o nga turanga U i roto i te whenu kotahi me te taurua. DNA me te whakaheke i te hua whakakaha 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 8.0), 150 mM NaCl, 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.
Mana Kounga
1.SDS-PAGE te parakore electrophoretic nui ake i te 98%
2.Te tairongo whakakaha, te mana o te puranga-ki-te-puranga, te pumau
3.I muri i te maimoatanga o te 1U o te UNG i te 50 ℃ mo te 2 meneti, ko te tauira kei roto U kei raro iho i te 103 kape me tino whakahekehia, kaore e taea te whakaputa hua whakakaha.
4.Karekau he mahi karihi o waho
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 |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 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.Ka taea te whakamahi i te UNG enzyme ki te tango i nga hua whakakaha dUTP kua poke mai i te punaha tauhohenga i mua i te tauhohenga whakakaha PCR, katahi ka karo i nga hua pai-tika na te poke o te hua.
2.Ko te pāmahana tino pai mo te UNG enzyme ki te whakamahi i roto i te tauhohenga PCR anti-contamination ko te tikanga 50 ℃ mo te 2mins;Ko te 95 ℃ te ahua o te whakakore mo te 5mins.
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.
