Okpomọkụ UNG nwere mmetụta
Okpomọkụ Sensitive UNG (TS-UNG) bụ enwetara site recombinant okwu na E. coli.Enzyme ahụ na-eme ka ntọhapụ nke uracil n'efu site na DNA nwere otu na nke nwere okpukpu abụọ ma ọ naghị arụ ọrụ megide RNA.E jiri ya tụnyere UNG enzyme nke E. coli gene origin, TS-UNG enzyme nwere ọrụ dị elu na okpomọkụ dị ala (20 ℃ ~ 37 ℃) ma na-enwe mmetụta okpomọkụ na ngwa ngwa adịghị arụ ọrụ (50 ℃), na-ezere mmebi nke dUTP nwere nkwalite. ngwaahịa n'ime ụlọ okpomọkụ site na arụ ọrụ fọdụrụnụ nwere ike ịdịgide mgbe arụghị ọrụ nke enzyme UNG.Ya mere, enzyme TS-UNG abụghị naanị dabara maka mmeghachi omume mgbochi mmetọ PCR, mana ọ dabara nke ọma na mmemme nkwalite RT-PCR ma enwere ike iji ya na mmeghachi omume mgbochi mmetọ nke RT-PCR.
Ngwa akwadoro
Mgbochi Mgbochi Mmeri
Ọnọdụ Nchekwa
-20ºC maka nchekwa ogologo oge, ekwesịrị ịgwakọta nke ọma tupu ejiri ya, zere ịza oyi mgbe niile.
Nchekwa nchekwa
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Nkọwa nkeji
Ọnụego enzyme achọrọ iji weda 1µg nke DNA nwere otu eriri nwere ntọala dU n'ime elekere 1 na 37 Celsius bụ otu ọrụ (U).
Njikwa ogo
1.SDS-PAGE electrophoretic dị ọcha karịa 98%
2.Ọrụ mmebi iwu, njikwa ogbe-na-batch, nkwụsi ike
3.Ọ dịghị ọrụ nuclease exogenous, ọ dịghị exogenous endonuclease ma ọ bụ exonuclease mmetọ.
Ntuziaka
| Ngwa | Olu (μL) | Ntinye uche ikpeazụ |
| 10 × PCR nchekwa (dNTP efu, Mg²+n'efu) | 5 | 1× |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (dochie dTTP) | - | 200-600 μM |
| 25 mmMgCl2 | 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 × Ngwakọta mbụa | 2 | 1× |
| Ụdị | - | 1 μg / mmeghachi omume |
| ddH₂O | Ruo 50 | - |
Mara: a: Ọ bụrụ na ejiri ya maka qPCR/qRT-PCR, a ga-agbakwunye nyocha nke fluorescent na sistemụ mmeghachi omume.Na-emekarị, njedebe ikpeazụ nke 0.2 μM nwere ike inye nsonaazụ dị mma;mgbe arụ ọrụ mmeghachi omume na-adịghị mma, enwere ike ịhazi ntinye nke primer na oke nke 0.2-1 μM.Ọtụtụ mgbe, a na-emezi mkpokọta nyocha n'ime oke 0.1-0.3 μM.Enwere ike ịme nnwale gradient ntinye uche iji chọta ngwakọta kacha mma nke primer na nyocha.
Ihe ndetu
1.The ezigbo mmeghachi omume okpomọkụ nke TS-UNG enzyme bụ dịtụ ala, na ọ nwere ike kachasị na nso nke 20 ℃ ~ 37 ℃, usoro onunu ogwu nke enzyme na mmeghachi omume oge nwere ike kachasị na nso nke 0.1 ~ 0.5 U, 5 ~ Nkeji 10;na enwere ike ime ka enzyme ahụ ghara ịrụ ọrụ na usoro ntụgharị ntụgharị.
2.Ọ dabara maka PCR na RT-PCR iji gbochie mmetọ.
3.Zere na-agbaze na friji ugboro ugboro, ma ghara ikpughe na nnukwu mgbanwe okpomọkụ.
4.Mkpụrụ ndụ ihe nketa dị iche iche a ga-amụba nwere arụmọrụ dị iche iche nke dUTP na uche na UNG enzyme, ya mere, ọ bụrụ na iji usoro UNG na-eduga n'ịbelata nchọpụta nchọpụta, a ghaghị imezi usoro mmeghachi omume na kachasị, ọ bụrụ na ịchọrọ nkwado teknụzụ, biko kpọtụrụ. ụlọ ọrụ anyị.
