Uracil ADN Glycosylase
Uracil-ADN Glycosylase (UNG cyangwa UDG) ni clone ya recombinant ya E.coli ifite uburemere bwa molekile 25 kDa.Ihagarika irekurwa rya uracil yubusa muri uracil irimo ADN imwe imwe kandi ifite imirongo ibiri, kandi ntigikora kurwanya RNA, kandi irashobora gukoreshwa mukurinda kwanduza ibicuruzwa byongera PCR.Ihame ryibikorwa rishingiye ku kuba niba dUTP isimbuwe na dTTP mu myitwarire ya PCR hanyuma hagashyirwaho ibicuruzwa byongera ingufu za PCR birimo ishingiro rya dU, enzyme irashobora guhitamo guca glycosidic ihuza ibice bya U mu murongo umwe kandi uhagaritse kabiri. ADN no gutesha agaciro ibicuruzwa byongera PCR.
Gusabwa
Kwiyongera kwanduye
Imiterere y'Ububiko
-20 ° C kubikwa igihe kirekire, bigomba kuvangwa neza mbere yo kubikoresha, irinde gukonjesha kenshi.
Ububiko
20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Igisobanuro
Ingano ya enzyme isabwa kugirango itesha agaciro 1µg ya ADN imwe imwe irimo ibice bya dU mu isaha 1 kuri 37 ° C ni 1.
Kugenzura ubuziranenge
1.SDS-PAGE isuku ya electrophoreque irenga 98%
2.Amplification sensitivite, icyiciro-kuri-kugenzura, gutuza
3.Nyuma ya 1U ya UNG ivuwe kuri 50 ℃ kuri 2mins, inyandikorugero irimo U munsi ya kopi 103 igomba guteshwa agaciro rwose kandi nta bicuruzwa byongera imbaraga bishobora gukorwa.
4.Nta gikorwa cya nuclease
Amabwiriza
| Ibigize | Umubumbe (μL) | Kwibanda kwanyuma |
| 10 × Buffer ya PCR (dNTP kubuntu, Mg²+ubuntu) | 5 | 1 × |
| DUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| DUTP (gusimbuza dTTP) | - | 200-600 μM |
| 25 mM MgCl2 | 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 × Primer mixa | 2 | 1 × |
| Inyandikorugero | - | Μ 1μg / reaction |
| ddH₂O | Kuri 50 | - |
Icyitonderwa: a: Niba ikoreshwa kuri qPCR / qRT-PCR, iperereza rya fluorescent rigomba kongerwaho muri sisitemu yo kwitwara.Mubisanzwe, primer yibanze ya 0.2 μM irashobora gutanga ibisubizo byiza;mugihe imikorere ya reaction ari mibi, kwibanda kwa primer birashobora guhinduka murwego rwa 0.2-1 μM.Mubisanzwe, intumbero ya probe itezimbere murwego rwa 0.1-0.3 μM.Ubushakashatsi bwibanze bwibanze burashobora gukorwa kugirango ubone ibyiza bya primer na probe.
Inyandiko
1.Enzyme ya UNG irashobora gukoreshwa mugukuraho ibicuruzwa byongerewe imbaraga bya dUTP muri sisitemu yo kubyitwaramo mbere ya PCR amplification reaction, hanyuma kugirango wirinde ibisubizo byiza-bibi kubera kwanduza ibicuruzwa.
2.Ubushuhe bwiza kuri enzyme ya UNG izakoreshwa mukurwanya kwanduza PCR mubisanzwe ni 50 ℃ kuri 2mins;imiterere yo kudakora ni 95 ℃ kuri 5mins.
3.Irinde gukonjesha kenshi, kandi ntugaragaze ihindagurika ryinshi ry'ubushyuhe.
4.Ingirabuzimafatizo zitandukanye zigomba kongererwa imbaraga zifite uburyo butandukanye bwo gukoresha dUTP no kumva neza imisemburo ya UNG, kubwibyo, niba gukoresha sisitemu ya UNG biganisha ku kugabanuka kwimyumvire, sisitemu yo kubyitwaramo igomba guhinduka kandi ikanozwa, niba ukeneye inkunga ya tekiniki, nyamuneka hamagara isosiyete yacu.
