Ubushyuhe bukabije UNG
Ubushyuhe bukabije UNG (TS-UNG) ni ukubona imvugo ya recombinant muri E. coli.Enzyme itera irekurwa rya uracil yubusa muri uracil irimo ADN imwe-imwe-ebyiri kandi idakora kuri RNA.Ugereranije na enzyme isanzwe ya UNG ikomoka kuri E. coli gene, enzyme ya TS-UNG ifite ibikorwa byinshi mubushyuhe buke (20 ℃ ~ 37 ℃) kandi irumva ubushyuhe kandi idakora byoroshye (50 ℃), irinda kwangirika kwa dUTP irimo amplification. ibicuruzwa ku bushyuhe bwicyumba nigikorwa gisigaye gishobora kuguma nyuma yo kudakora enzyme isanzwe ya UNG.Kubwibyo, enzyme ya TS-UNG ntabwo ikwiranye gusa no gukumira kwanduza PCR gusa, ahubwo ihuye neza na gahunda yo kongera ingufu za RT-PCR kandi irashobora gukoreshwa muburyo bwo gukumira kwanduza RT-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 7.5), 100 mM NaCl, 0.1 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 mumasaha 1 kuri 37 ° C nigice 1 cyibikorwa (U).
Kugenzura ubuziranenge
1.SDS-PAGE isuku ya electrophoreque irenga 98%
2.Igikorwa cyo gutesha agaciro, kugenzura-icyiciro, kugenzura
3.Ntabwo ibikorwa bya nuclease bidasanzwe, nta endogenuclease ya exogenous cyangwa exonuclease yanduye.
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. |
| 1 U / μLTS-UNG | 0.5 (0.1-0.5) | 0.5 U (0.1-0.5U) |
| 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.Ubushyuhe bwiza bwa reaction ya TS-UNG enzyme iri hasi cyane, kandi irashobora gutezimbere murwego rwa 20 ℃ ~ 37 ℃, dosiye ya enzyme nigihe cyo kuyitwara irashobora gutezimbere murwego rwa 0.1 ~ 0.5 U, 5 ~ Iminota 10;na enzyme irashobora kudakora mugikorwa cyo kwandukura.
2.Birakwiye kuri PCR na RT-PCR kugirango birinde kwanduza.
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.
