Enzymes UDG/UNG
UDG (uracil DNA glycosylase) nwere ike ime ka hydrolysis nke njikọ N-glycosidic dị n'etiti uracil base na sugar-phosphate azụ na ssDNA na dsDNA.Ọ nwere ike ijikwa mmetọ ikuku aerosol n'ụzọ dị mfe ma dabara adaba maka sistemu ihe omimi ihe omimi dị ka PCR, qPCR, RT-qPCR na LAMP.
Nkọwapụta
| Onye ọbịa okwu | Recombinant E.coliwith uracil DNA glycosylase gene |
| Ibu molekụla | 24.8kDa |
| Ịdị ọcha | ≥95% (SDS-PAGE) |
| Ọkụ anaghị arụ ọrụ | 95 ℃, 5 ~ 10 nkeji |
| Nkọwa nkeji | A kọwapụtara otu nkeji (U) dị ka ọnụọgụ enzyme chọrọ iji kpalite hydrolysis nke l μg dU nwere dsDNA n'ime nkeji 30 na 25 ℃. |
Nchekwa
Ekwesịrị ịchekwa ngwaahịa ahụ na-25 ℃ ~ -15 Celsius C maka afọ abụọ.
Ntuziaka
1.Nkwadebe nke ngwakọta mmeghachi omume PCR dị ka usoro ndị a si dị
| Ngwa | Olu (μL) | Ntinye uche ikpeazụ |
| Ihe nchekwa 10 × PCR (Mg²+Plus) | 5 | 1× |
| 25 mmol / LMgCl | 3 | 1.5 mmol / L |
| dUTP (10 mmol / L) | 3 | 0.6 mmol / L |
| dCTP/dGTP/dATP/dTTP(10mmol/Leach) | 1 | 0.2 mmol / leach |
| DNA template | X | - |
| Primer1 (10μmol/L) | 2 | 0.4 μmol / L |
| Nke mbụ 2 (10μmol/L) | 2 | 0.4 μmol / L |
| Taq DNA Polymerase (5 U/μL) | 0.5 | 0.05 U/μL |
| Uracil DNA Glycosylase (UDG/UNG), 1 U/μL | 1 | 1 U/μL |
| ddH₂O | Ruo 50 | - |
Mara: Dị ka nnwale chọrọ, enwere ike gbanwee ntinye ikpeazụ nke dUTP n'etiti 0.2-0.6 mmol/L, na 0.2 mmol/L dTTP nwere ike ịgbakwunye nhọrọ.
2.Usoro mmụba
| Nzọụkwụ okirikiri | Okpomọkụ | Oge | okirikiri |
| dU nwere ndebiri ndebiri | 25 ℃ | Nkeji 10 | 1 |
| UDG ịgbalite, ndebiri denaturation mbụ | 95 ℃ | 5 ~ 10 nkeji | 1 |
| Denaturation | 95 ℃ | 10 sk |
30-35 |
| Na-ewe iwe | 60 ℃ | 20 sk | |
| Mgbatị | 72 ℃ | 30sk/kb | |
| Mgbatị ikpeazụ | 72 ℃ | 5 min | 1 |
Mara: Enwere ike ịhazi oge mmeghachi omume na 25 ° C n'ime 5-10mins dịka ihe nyocha chọrọ.
Ihe ndetu
1.UDG na-arụsi ọrụ ike n'ọtụtụ ebe nchekwa PCR.
2.Ekwesịrị ịchekwa enzymes n'ime igbe ice ma ọ bụ na mmiri ịsa mmiri mgbe ejiri ya, a ga-echekwa ya na-20 Celsius C ozugbo ejiri ya.
3.Biko yiri PPE dị mkpa, uwe ụlọ nyocha na uwe aka, iji hụ na ahụike na nchekwa gị!
