Uracil DNA Glycosylase
I-Uracil-DNA Glycosylase (UNG noma i-UDG) iyinhlanganisela ye-E.coli enesisindo samangqamuzana angama-25 kDa.Igqugquzela ukukhishwa kwe-uracil yamahhala ku-uracil equkethe i-DNA enemicu eyodwa nephindwe kabili, futhi ayisebenzi ngokumelene ne-RNA, futhi ingasetshenziswa ukuvimbela ukungcoliswa kwemikhiqizo yokukhulisa i-PCR.Umgomo wesenzo usekelwe eqinisweni lokuthi uma i-dUTP ithathelwa indawo i-dTTP ekuphenduleni kwe-PCR futhi kwakheka umkhiqizo wokukhulisa i-PCR oqukethe izisekelo ze-dU, i-enzyme inganqamula ngokukhetha isibopho se-glycosidic sezisekelo ze-U kumugqa owodwa futhi ophindwe kabili. I-DNA futhi yehlise isithunzi umkhiqizo wokukhulisa i-PCR.
Isicelo Esinconyiwe
Ukukhulisa Ukuvimbela Ukungcola
Isimo Sesitoreji
-20°C ukuze igcinwe isikhathi eside, kufanele ixutshwe kahle ngaphambi kokusetshenziswa, gwema ukuncibilika kweqhwa njalo.
Ibhafa yesitoreji
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Incazelo Yeyunithi
Inani le-enzyme elidingekayo ukuze kwehliswe i-1µg ye-DNA enomucu owodwa equkethe izisekelo ze-dU ngehora elingu-1 ku-37°C yiyunithi engu-1.
Ikhwalithi yokulawula
1.Ukuhlanzeka kwe-electrophoretic ye-SDS-PAGE okukhulu kuno-98%
2.Ukuzwela kokukhulisa, ukulawula kwe-batch-to-batch, ukuzinza
3.Ngemuva kokuthi i-1U ye-UNG ilashwe ku-50℃ imizuzu emi-2, isifanekiso esiqukethe u-U ngezansi kwamakhophi ayi-103 kufanele sehliswe ngokuphelele futhi awukho umkhiqizo wokukhulisa amandla ongakhiqizwa.
4.Awukho umsebenzi we-nuclease exogenous
Iziyalezo
| Izingxenye | Ivolumu (μL) | Ukugxila kokugcina |
| 10 × PCR Buffer (dNTP mahhala, Mg²+khulula) | 5 | 1× |
| I-dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| I-dUTP (buyisela i-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 × I-Primer Mixa | 2 | 1× |
| Isifanekiso | - | ＜1μg/ukusabela |
| ddH₂O | Kuze ku-50 | - |
Qaphela: a: Uma isetshenziselwa i-qPCR/qRT-PCR, i-fluorescent probe kufanele yengezwe ohlelweni lokusabela.Ngokuvamile, ukuhlushwa kwe-primer yokugcina ye-0.2 μM kunganikeza imiphumela emihle;lapho ukusebenza kokusabela kukubi, ukugxilwa kwe-primer kungalungiswa ebangeni lika-0.2-1 μM.Ngokuvamile, ukugxiliswa kwe-probe kulungiselelwa ku-0.1-0.3 μM.Ukuhlolwa kwe-concentration gradient kungenziwa ukuze kutholwe inhlanganisela engcono kakhulu ye-primer ne-probe.
Amanothi
1.I-enzyme ye-UNG ingasetshenziswa ukususa imikhiqizo yokukhulisa i-dUTP engcolile ohlelweni lokusabela ngaphambi kokusabela kokukhulisa i-PCR, bese kugwenywa imiphumela engemihle ngenxa yokungcoliswa komkhiqizo.
2.Izinga lokushisa elilungile le-enzyme ye-UNG okufanele lisetshenziswe ekuphenduleni kwe-PCR elwa nokungcoliswa ngokuvamile lingu-50℃ imizuzu emi-2;isimo sokungasebenzi singu-95℃ imizuzu emi-5.
3.Gwema ukuncibilika okuvamisile, futhi ungabeki obala ekushintshashintsheni okukhulu kwezinga lokushisa.
4.Izakhi zofuzo ezihlukene okufanele zikhuliswe zinokusebenza okuhlukile kokusetshenziswa kwe-dUTP kanye nokuzwela kwe-enzyme ye-UNG, ngakho-ke, uma ukusetshenziswa kohlelo lwe-UNG kuholela ekwehleni kokuzwela kokutholwa, uhlelo lokusabela kufanele lulungiswe futhi lwenziwe kahle, uma udinga ukwesekwa kobuchwepheshe, sicela uthinte. inkampani yethu.
