Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG ma ọ bụ UDG) bụ ihe na-emegharị emegharị nke E.coli nwere ịdị arọ molekụla nke 25 kDa.Ọ na-eme ka ntọhapụ nke uracil n'efu site na uracil nwere otu eriri na DNA nke nwere eriri abụọ, ọ naghị arụ ọrụ megide RNA, enwere ike iji ya gbochie mmetọ nke ngwaahịa nkwalite PCR.Ụkpụrụ nke ime ihe dabere n'eziokwu ahụ bụ na ọ bụrụ na ejiri dUTP dochie anya dTTP na mmeghachi omume PCR na mmepụta PCR nke nwere dU bases na-emepụta, enzyme ahụ nwere ike ịhọrọ imebi njikọ glycosidic nke U bases na otu-stranded na okpukpu abụọ. DNA ma weda ngwaahịa nkwalite 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 8.0), 150 mM NaCl, 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 nkeji.
Njikwa ogo
1.SDS-PAGE electrophoretic dị ọcha karịa 98%
2.Mmetụta mmụba, njikwa batch-to-batch, nkwụsi ike
3.Mgbe a gwọchara 1U nke UNG na 50℃ maka nkeji abụọ, template nwere U n'okpuru 103 ga-emebi kpamkpam na enweghị ike ịmepụta ngwaahịa nkwalite.
4.Enweghị ọrụ nuclease dịpụrụ adịpụ
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 |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 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 n'ime usoro 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.Enwere ike iji UNG enzyme wepụ ngwaahịa nkwalite dUTP emetọghị na sistemu mmeghachi omume tupu mmeghachi omume mmụba PCR, wee zere nsonaazụ adịgboroja n'ihi mmetọ ngwaahịa.
2.Okpomọkụ kachasị mma maka enzyme UNG nke a ga-eji na mmeghachi omume PCR na-egbochi mmetọ bụ 50 ℃ maka nkeji abụọ;Ọnọdụ arụghị ọrụ bụ 95 ℃ maka 5mins.
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.
