Li-enzyme tsa UDG/UNG
UDG(uracil DNA glycosylase) e ka etsa hore ho be le hydrolysis ea khokahanyo ea N-glycosidic pakeng tsa motheo oa uracil le mokokotlo oa tsoekere-phosphate ho ssDNA le dsDNA.E ka laola tšilafalo ea aerosol habonolo 'me e loketse litsamaiso tse tloaelehileng tsa baeloji ea limolek'hule tse kang PCR, qPCR, RT-qPCR le LAMP.
Litlhaloso
| Expression Host | Recombinant E. coliwith uracil DNA glycosylase gene |
| Boima ba Molek'hule | 24. 8kDa |
| Bohloeki | ≥95% (SDS-PAGE) |
| Ho se sebetse ha mocheso | 95 ℃, 5 ~ 10 mets |
| Tlhaloso ea Yuniti | Yuniti e le 'ngoe (U) e hlalosoa e le palo ea enzyme e hlokahalang ho etsa hore hydrolysis ea l μg dU e nang le dsDNA ka metsotso e 30 ho 25 ℃. |
Polokelo
Sehlahisoa se lokela ho bolokoa ho -25 ℃ ~ -15 ° C ka lilemo tse peli.
Litaelo
1.Ho lokisoa ha motsoako oa karabelo ea PCR ho latela sistimi e latelang
| Likaroloana | Bolumo (μL) | Khatiso ea ho qetela |
| 10×PCR Buffer (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 ea Setšoantšo | X | - |
| Primer1 (10μmol/L) | 2 | 0.4 μmol/L |
| Primer 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 | Ho fihlela ho 50 | - |
Hlokomela: Ho latela litlhoko tsa liteko, khatello ea ho qetela ea dUTP e ka fetoloa lipakeng tsa 0.2-0.6 mmol/L, le 0.2 mmol/L dTTP e ka eketsoa ka boikhethelo.
2.Mokhoa oa ho ntlafatsa
| Mohato oa potoloho | Mocheso | Nako | Lipotoloho |
| Ho senyeha ha template e nang le dU | 25 ℃ | 10metsotso | 1 |
| UDG activation, denaturation ea pele ea template | 95℃ | 5-10metsotso | 1 |
| Denaturation | 95℃ | 10 sec |
30-35 |
| Annealing | 60 ℃ | 20 metsotsoana | |
| Katoloso | 72℃ | 30sec/kb | |
| Katoloso ea ho qetela | 72℃ | 5 mets | 1 |
Hlokomela: Nako ea karabelo ho 25 ° C e ka fetoloa nakong ea 5-10mins ho latela litlhoko tsa liteko.
Lintlha
1.UDG e sebetsa ho li-buffers tse ngata tsa PCR.
2.Li-enzyme li lokela ho bolokoa ka lebokoseng la leqhoa kapa ka bateng ea leqhoa ha li sebelisoa, 'me li lokela ho bolokoa ho -20 ° C hang ka mor'a ho sebelisoa.
3.Ka kopo, roala PPE e hlokahalang, jase e joalo ea lab le liatlana, ho netefatsa bophelo bo botle le polokeho ea hau!
