Ubushushu Bunovakalelo UNG
Iqondo lobushushu UNG (TS-UNG) lifunyenwe ngokuphinda kubonakaliswe kwi-E. coli.I-enzyme ibangela ukukhululwa kwe-uracil yamahhala kwi-uracil equkethe i-DNA enye kunye ne-double-stranded DNA kwaye ayisebenzi ngokuchasene ne-RNA.Xa kuthelekiswa ne-enzyme yesiqhelo ye-UNG yemvelaphi yemfuza ye-E. coli, i-enzyme ye-TS-UNG inomsebenzi ophezulu kumaqondo obushushu aphantsi (20℃ ~ 37℃) kwaye inochuku kubushushu kwaye ingasebenzi ngokulula (50℃), inqanda ukuthotywa kwe-dUTP-equlathe ukukhulisa iimveliso kubushushu begumbi ngumsebenzi oshiyekileyo onokuthi uhlale emva kokungasebenzi kwe-enzyme yesiqhelo ye-UNG.Ke ngoko, i-enzyme ye-TS-UNG ayifanelekanga kuphela kwi-PCR yokuthintela ukungcoliseka, kodwa ikwahambelana kakuhle nenkqubo yokukhulisa i-RT-PCR kwaye ingasetyenziswa kwi-RT-PCR yokuthintela ukuchaphazeleka.
Isicelo esicetyiswayo
Ukwandiswa koThintelo loNgcoliseko
Imeko yoGcino
-20°C kwindawo yokugcina ixesha elide, kufuneka ixutywe kakuhle phambi kokusetyenziswa, kunqande ukunyibilika komkhenkce rhoqo.
Isithinteli sogcino
20 mM Tris-HCl (pH 7.5) , 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Inkcazelo yeyunithi
Ubungakanani be-enzyme efunekayo ukuthobisa i-1µg ye-DNA enemisonto enye equlethe iziseko ze-dU kwiyure ye-1 kwi-37 ° C yiyunithi ye-1 yomsebenzi (U).
Ulawulo lwemeko
1.I-SDS-PAGE ukucoceka kwe-electrophoretic ngaphezulu kwe-98%
2.Umsebenzi wokuthotywa, ukulawula i-batch-to-batch, ukuzinza
3.Akukho msebenzi we-nuclease exogenous, akukho endonuclease exogenous okanye exonuclease ungcoliseko.
Imiyalelo
| Amacandelo | Umthamo (μL) | Ugxininiso lokugqibela |
| 10 × PCR Buffer (dNTP simahla, Mg²+simahla) | 5 | 1× |
| ii-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/impendulo |
| ddH₂O | Ukuya kwi50 | - |
Qaphela: a: Ukuba isetyenziselwa i-qPCR/qRT-PCR, iprobe yefluorescent kufuneka yongezwe kwindlela yokusabela.Ngokuqhelekileyo, i-primer concentration yokugqibela ye-0.2 μM inokunika iziphumo ezilungileyo;xa i-reaction performance ihlwempuzekile, i-primer concentration inokulungiswa kuluhlu lwe-0.2-1 μM.Ngokuqhelekileyo, i-probe concentration ilungiselelwe kuluhlu lwe-0.1-0.3 μM.Imifuniselo yegradient yoxinaniso inokwenziwa ukufumana eyona ndibaniselwano yeprimer kunye neprobe.
Amanqaku
1.Ubushushu obufanelekileyo bokusabela kwe-enzyme ye-TS-UNG iphantsi ngokwentelekiso, kwaye inokuphuculwa kuluhlu lwe-20℃ ~ 37℃, idosi ye-enzyme kunye nexesha lokuphendula linokuphuculwa kuluhlu lwe-0.1 ~ 0.5 U, 5~ Imizuzu eyi-10;kwaye i-enzyme inokuthi ingasebenzi kwinkqubo yokubhala umva.
2.Ifanelekile kwi-PCR kunye ne-RT-PCR ukuthintela ukungcoliseka.
3.Kuphephe ukunyibilika komkhenkce rhoqo, kwaye ungabi sesichengeni kuguquko olukhulu lobushushu.
4.Izakhi zofuzo ezahlukeneyo eziza kwandiswa zineendlela ezahlukeneyo zokusetyenziswa kwe-dUTP kunye nobuntununtunu kwi-enzyme ye-UNG, ke ngoko, ukuba ukusetyenziswa kwenkqubo ye-UNG kukhokelela ekunciphiseni uvakalelo lokubona, inkqubo yokusabela kufuneka ihlengahlengiswe kwaye iphuculwe, ukuba ufuna inkxaso yobugcisa, nceda uqhagamshelane. inkampani yethu.
