Izinga lokushisa liyazwela UNG
I-Temperature Sensitive UNG (TS-UNG) iwuphawu olutholwa kabusha nge-E. coli.I-enzyme igqugquzela ukukhishwa kwe-uracil yamahhala ku-uracil equkethe i-DNA eyodwa enemicu emibili futhi ayisebenzi ngokumelene ne-RNA.Uma kuqhathaniswa ne-enzyme evamile ye-UNG yemvelaphi yofuzo lwe-E. coli, i-enzyme ye-TS-UNG inomsebenzi ophezulu emazingeni okushisa aphansi (20℃~37℃) futhi izwela izinga lokushisa futhi ayisebenzi kalula (50℃), igwema ukuwohloka kokukhuliswa okuqukethe i-dUTP. imikhiqizo emazingeni okushisa asekamelweni ngomsebenzi osele ongasala ngemuva kokungasebenzi kwe-enzyme evamile ye-UNG.Ngakho-ke, i-enzyme ye-TS-UNG ayifanele nje kuphela ukusabela kokuvimbela ukungcoliswa kwe-PCR, kodwa futhi ihambisana kahle nohlelo lokukhulisa i-RT-PCR futhi ingasetshenziswa ekuphenduleni kokuvimbela ukungcoliswa kwe-RT-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 7.5) , 100 mM NaCl, 0.1 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 iyunithi engu-1 yomsebenzi (U).
Ikhwalithi yokulawula
1.Ukuhlanzeka kwe-electrophoretic ye-SDS-PAGE okukhulu kuno-98%
2.Umsebenzi wokucekela phansi, ukulawulwa kwe-batch-to-batch, ukuzinza
3.Awukho umsebenzi we-nuclease exogenous, akukho endonuclease exogenous noma exonuclease ukungcola.
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 |
| 1 U/μLTS-UNG | 0.5 (0.1-0.5) | 0.5 U (0.1-0.5U) |
| 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.Izinga lokushisa elilungile lokusabela le-enzyme ye-TS-UNG liphansi uma kuqhathaniswa, futhi lingenziwa kahle ebangeni lika-20℃~37℃, umthamo we-enzyme nesikhathi sokuphendula singenziwa kahle ku-0.1~0.5 U, 5~ 10 imiz;futhi i-enzayimu ingenziwa ingasebenzi enqubweni yokulotshwa okuhlanekezelwe.
2.Ifanele i-PCR kanye ne-RT-PCR ukuvimbela ukungcola.
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.
