UDG / UNG Enzymes
UDG (uracil ADN glycosylase) irashobora guhagarika hydrolysis ya N-glycosidic ihuza urwego rwa uracil hamwe nisukari-fosifate yumugongo muri ssDNA na dsDNA.Irashobora kurwanya byoroshye umwanda wa aerosol kandi ikwiranye na sisitemu ya biologiya isanzwe nka PCR, qPCR, RT-qPCR na LAMP.
Ibisobanuro
| Ikirangantego | Recombinant E. coliwith uracil ADN glycosylase gene |
| Uburemere bwa molekile | 24. 8kDa |
| Isuku | ≥95% (SDS-PAGE) |
| Gushyushya | 95 ℃, 5 ~ 10 min |
| Igisobanuro | Igice kimwe (U) gisobanurwa nkubunini bwa enzyme isabwa guhagarika hydrolysis ya l μg dU irimo dsDNA muminota 30 kuri 25 ℃. |
Ububiko
Ibicuruzwa bigomba kubikwa kuri-25 ℃ ~ -15 ° C kumyaka ibiri.
Amabwiriza
1.Gutegura kuvanga PCR bivanze ukurikije sisitemu ikurikira
| Ibigize | Umubumbe (μL) | Kwibanda kwanyuma |
| 10 × Buffer ya 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 / Kureka |
| Icyitegererezo ADN | X | - |
| Primer1 (10μmol / L) | 2 | 0.4 μ mol / L. |
| Primer 2 (10μmol / L) | 2 | 0.4 μ mol / L. |
| Taq ADN Polymerase (5 U / μL) | 0. 5 | 0. 05 U / μL |
| Uracil ADN Glycosylase (UDG / UNG), 1 U / μL | 1 | 1 U / μL |
| ddH₂O | Kugera kuri 50 | - |
Icyitonderwa: Ukurikije ibisabwa byubushakashatsi, intumbero yanyuma ya dUTP irashobora guhinduka hagati ya 0.2-0,6 mmol / L, na 0.2 mmol / L dTTP irashobora kongerwaho guhitamo.
2.Uburyo bwo kongera imbaraga
| Intambwe | Ubushyuhe | Igihe | Amagare |
| dU irimo inyandikorugero yo gutesha agaciro | 25 ℃ | 10min | 1 |
| Gukora UDG, icyitegererezo cyambere cyo gutandukana | 95 ℃ | 5 ~ 10min | 1 |
| Gutandukana | 95 ℃ | Amasegonda 10 |
30-35 |
| Annealing | 60 ℃ | 20 amasegonda | |
| Kwagura | 72 ℃ | 30sec / kb | |
| Kwagura kwa nyuma | 72 ℃ | Imin | 1 |
Icyitonderwa: Igihe cyo kwitwara kuri 25 ° C kirashobora guhinduka muminota 5-10m ukurikije ibisabwa mubushakashatsi.
Inyandiko
1.UDG ikora mubikorwa byinshi bya PCR reaction.
2.Enzymes igomba kubikwa muri icebox cyangwa ku bwogero bwa barafu iyo ikoreshejwe, kandi igomba kubikwa kuri 20 ° C ako kanya nyuma yo kuyikoresha.
3.Nyamuneka nyamuneka wambare PPE ikenewe, ikote rya laboratoire hamwe na gants, kugirango ubuzima bwawe n'umutekano wawe!
