Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG los yog UDG) yog ib tug recombinant clone ntawm E.coli nrog ib tug molecular hnyav ntawm 25 kDa.Nws catalyzes tso tawm dawb uracil los ntawm uracil-muaj ib leeg-stranded thiab ob-stranded DNA, thiab yog inactive tiv thaiv RNA, thiab yuav siv tau los tiv thaiv kev kis ntawm PCR amplification khoom.Lub hauv paus ntsiab lus ntawm kev nqis tes ua yog raws li qhov tseeb tias yog tias dUTP hloov pauv rau dTTP hauv cov tshuaj tiv thaiv PCR thiab PCR amplification khoom uas muaj dU bases yog tsim, lub enzyme tuaj yeem xaiv ua txhaum cov glycosidic daim ntawv cog lus ntawm U hauv paus hauv ib leeg-stranded thiab ob-stranded. DNA thiab degrade PCR amplification khoom.
Pom zoo daim ntawv thov
Kev Tiv Thaiv Kev Tiv Thaiv Kev Tshaj Tawm
Kev cia khoom
-20 ° C rau lub sij hawm ntev cia, yuav tsum tau tov zoo ua ntej siv, tsis txhob nquag khov-thaw.
Cia tsis
20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Unit txhais
Tus nqi ntawm enzyme yuav tsum degrade 1µg ntawm ib leeg-stranded DNA uas muaj dU bases nyob rau hauv 1 teev ntawm 37 ° C yog 1 unit.
Kev Tswj Xyuas Zoo
1.SDS-PAGE electrophoretic purity ntau dua 98%
2.Amplification rhiab heev, batch-to-batch tswj, stability
3.Tom qab 1U ntawm UNG raug kho ntawm 50 ℃ rau 2mins, cov qauv uas muaj U hauv qab 103 daim ntawv yuav tsum tau degraded tag nrho thiab tsis muaj amplification khoom yuav ua tau.
4.Tsis muaj exogenous nuclease ua haujlwm
Cov lus qhia
| Cheebtsam | Ntim (μL) | Thaum kawg concentration |
| 10 × PCR Buffer (dNTP dawb, Mg²+dawb) | 5 | 1 × |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (hloov dTTP) | - | 200-600 hli |
| 25 mMgcl2 | 2 - 8 L | 1-4 mM |
| 5 U / μL Taq | 0.25 | 1.25 ua |
| 5 U / μL UNG | 0.25 (0.1-0.5 hli) | 0.25 U (0.1-0.5 hli) |
| 25 × Primer Mixib | 2 | 1 × |
| Template | - | ＜1μg / tshuaj |
| ddH₂O | Rau 50 | - |
Nco tseg: a: Yog siv rau qPCR/qRT-PCR, fluorescent sojntsuam yuav tsum tau ntxiv rau hauv cov tshuaj tiv thaiv.Feem ntau, qhov kawg primer concentration ntawm 0.2 μM tuaj yeem muab cov txiaj ntsig zoo;Thaum cov tshuaj tiv thaiv kev ua haujlwm tsis zoo, cov primer concentration tuaj yeem hloov kho hauv thaj tsam ntawm 0.2-1 μM.Feem ntau, kev sojntsuam concentration yog optimized nyob rau hauv thaj tsam ntawm 0.1-0.3 μM.Concentration gradient thwmsim tuaj yeem ua tau los nrhiav qhov zoo tshaj plaws ua ke ntawm primer thiab sojntsuam.
Sau ntawv
1.UNG enzyme tuaj yeem siv los tshem tawm cov kab mob dUTP amplification khoom los ntawm cov tshuaj tiv thaiv kab mob ua ntej PCR amplification cov tshuaj tiv thaiv, tom qab ntawd kom tsis txhob muaj cov txiaj ntsig tsis zoo vim cov khoom sib kis.
2.Qhov ntsuas kub zoo rau UNG enzyme yuav tsum tau siv rau hauv cov tshuaj tiv thaiv kab mob PCR feem ntau yog 50 ℃ rau 2mins;Cov mob inactivation yog 95 ℃ rau 5 mins.
3.Tsis txhob nquag khov-thaw, thiab tsis txhob cuam tshuam rau qhov kub hloov pauv loj.
4.Cov noob sib txawv yuav tsum tau nthuav dav muaj qhov sib txawv ntawm kev siv dUTP thiab rhiab heev rau UNG enzyme, yog li ntawd, yog tias kev siv UNG system ua rau txo qis hauv kev tshawb pom, cov tshuaj tiv thaiv yuav tsum tau kho thiab kho kom zoo, yog tias koj xav tau kev txhawb nqa, thov hu rau peb lub tuam txhab.
