Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG ama UDG) waa isku-dhafan ee E.coli oo miisaankeedu yahay 25 kDa.Waxa ay kicisaa sii daynta uracilka bilaashka ah ee uracil ka kooban DNA hal xadhig ah iyo laba-geesood ah, waana mid aan firfircoonayn oo lid ku ah RNA, waxaana loo isticmaali karaa si looga hortago faddaraynta alaabta kor u qaadida PCR.Mabda'a ficilku wuxuu ku salaysan yahay xaqiiqda ah in haddii dUTP lagu beddelo dTTP falcelinta PCR iyo wax soo saarka PCR-ga oo ka kooban saldhigyada dU la sameeyay, ensaymku wuxuu si xushmad leh u jebin karaa curaarta glycosidic ee saldhigyada U ee hal-xajin iyo laba-jibbaaran. DNA oo hoos u dhiga badeecada kor u qaadida PCR.
Codsiga lagu taliyay
Kordhinta Ka-hortagga Wasakhda
Xaalada Kaydinta
-20°C kaydinta muddada dheer, waa in si fiican loo qaso ka hor inta aan la isticmaalin, iska ilaali baraf-dhalaalid joogto ah.
Kaydinta kaydinta
20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, xasiliye, 50% Glycerol.
Qeexitaan Unug
Qadarka ensaymka loo baahan yahay si loo dhimo 1µg ee DNA-da-geeska ah ee ka kooban saldhigyada dU 1 saac 37°C waa 1 unug.
Xakamaynta tayada
1.SDS-PAGE nadiifinta korantada ee ka weyn 98%
2.Dareenka weynaynta, xakamaynta dufcaddii, xasilloonida
3.Ka dib markii 1U ee UNG lagu daweeyo 50℃ 2mins, template ka kooban U ee ka hooseeya 103 koobi waa in gabi ahaanba la hoos u dhigaa oo aan la soo saari karin badeeco kordhin
4.Ma jiro hawl nuklease ah oo dibadda ka ah
Tilmaamaha
| Qaybaha | Mugga (μL) | U fiirsashada kama dambaysta ah |
| 10 × PCR Buffer (dNTP bilaash ah, Mg²+bilaash) | 5 | 1× |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (ku beddel 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 × Isku dhafka koowaada | 2 | 1× |
| Template | - | 1 μg/ falcelin |
| ddH₂O | Ilaa 50 | - |
Fiiro gaar ah: a: Haddii loo isticmaalo qPCR/qRT-PCR, baaritaanka fluorescent waa in lagu daraa habka falcelinta.Caadi ahaan, fiirsashada ugu dambeysa ee 0.2 μM waxay ku siin kartaa natiijooyin wanaagsan;marka waxqabadka falcelinta uu liito, diirada asaasiga ah waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-1 μM.Caadi ahaan, xoogga saarista baaritaanka waxaa lagu hagaajiyaa inta u dhexeysa 0.1-0.3 μM.Tijaabooyin isugeyn xoog leh ayaa la sameyn karaa si loo helo isku dhafka ugu wanaagsan ee asaasiga ah iyo baaritaanka.
Xusuusin
1.Enzyme UNG waxaa loo isticmaali karaa in laga saaro alaabada kordhinta dUTP ee wasakhaysan nidaamka falcelinta ka hor falcelinta kordhinta PCR, ka dib si looga fogaado natiijooyin been abuur ah oo ay ugu wacan tahay wasakheynta alaabta.
2.Heerkulka ugu fiican ee enzyme UNG ee loo isticmaalo falcelinta PCR-ka-hortagga faddaraynta guud ahaan waa 50℃ 2mins;xaalada shaqa la'aantu waa 95 ℃ 5mins.
3.Iska ilaali baraf-dhalaalid oo joogto ah, hana u bandhigin isbedbeddelka heerkulka weyn.
4.Hidde-sidayaasha kala duwan ee la xoojiyey waxay leeyihiin waxtarka isticmaalka kala duwan ee dUTP iyo dareenka UNG enzyme, sidaas darteed, haddii isticmaalka nidaamka UNG uu keeno hoos u dhaca dareenka ogaanshaha, nidaamka falcelinta waa in la hagaajiyaa oo la hagaajiyaa, haddii aad u baahan tahay taageero farsamo, fadlan la xiriir. shirkadeena.
