UDG/UNG Enzymes
UDG (uracil DNA glycosylase) waxay kicin kartaa hydrolysis ee isku xirka N-glycosidic ee u dhexeeya saldhigga uracil iyo laf dhabarta sonkorta-fosfate ee ssDNA iyo dsDNA.Waxay si fudud u xakameyn kartaa wasakhowga hawada waxayna ku habboon tahay nidaamyada bayoolojiga molecular ee caadiga ah sida PCR, qPCR, RT-qPCR iyo LAMP.
Tilmaamaha
| Martigaliyaha Hadalka | Dib-u-habaynta E. koliwith uracil DNA glycosylase hidda-wadaha |
| Miisaanka Molecular | 24. 8kDa |
| daahirnimo | ≥95% (SDS-PAGE) |
| Dhaqdhaqaaq la'aanta kulaylka | 95℃, 5 ~ 10 daqiiqo |
| Qeexitaan Unug | Hal unug (U) ayaa lagu qeexaa inay tahay cadadka ensaymka ee loo baahan yahay si loo kiciyo hydrolysis of l μg dU-ka kooban dsDNA gudaha 30 daqiiqo at 25℃. |
Kaydinta
Alaabta waa in lagu kaydiyaa at-25 ℃ ~ -15 ° C laba sano.
Tilmaamaha
1.Diyaarinta isku dhafka falcelinta PCR iyadoo la raacayo nidaamka soo socda
| Qaybaha | Mugga (μL) | U fiirsashada kama dambaysta ah |
| 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 |
| Qaabka DNA-da | X | - |
| Primer1 (10μmol/L) | 2 | 0.4 μmol/L |
| 10 μmol/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 | Ilaa 50 | - |
Fiiro gaar ah: Marka loo eego shuruudaha tijaabada, xoojinta ugu dambeysa ee dUTP waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-0.6 mmol/L, iyo 0.2 mmol/L dTTP ayaa lagu dari karaa si door ah.
2.Habka kor u qaadida
| Tallaabo wareeg | Heerkulka | Waqtiga | Wareegtada |
| Duuminta template du-ka kooban | 25℃ | 10 daqiiqo | 1 |
| Firfircoonida UDG, qaabaynta denaturation bilowga ah | 95℃ | 5 ~ 10 daqiiqo | 1 |
| Denaturation | 95℃ | 10 ilbiriqsi |
30-35 |
| Annealing | 60℃ | 20 ilbiriqsi | |
| Kordhinta | 72℃ | 30 ilbiriqsi/kb | |
| Kordhinta kama dambaysta ah | 72℃ | 5 daqiiqo | 1 |
Fiiro gaar ah: Waqtiga falcelinta ee 25°C waxaa lagu hagaajin karaa 5-10mins gudahood iyadoo loo eegayo shuruudaha tijaabada.
Xusuusin
1.UDG waxay ku firfircoon tahay badiyaa difaacyada falcelinta PCR.
2.Enzymes waa in lagu kaydiyaa sanduuqa barafka ama qubeyska barafka marka la isticmaalo, waana in lagu kaydiyaa at-20°C isla markiiba ka dib isticmaalka.
3.Fadlan xidho PPE-ga lagama maarmaanka ah, sida jaakada shaybaadhka iyo galoofyada, si loo hubiyo caafimaadkaaga iyo badbaadadaada!
