Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG kapena UDG) ndi chophatikizanso cha E.coli chokhala ndi molekyulu yolemera 25 kDa.Imathandizira kutulutsidwa kwa uracil waulere kuchokera ku uracil wokhala ndi DNA imodzi yokhala ndi zingwe ziwiri, ndipo imakhala yosagwira ntchito motsutsana ndi RNA, ndipo ingagwiritsidwe ntchito kuteteza kuipitsidwa kwa zinthu zokulitsa za PCR.Mfundo yochitirapo kanthu imachokera pa mfundo yakuti ngati dUTP ilowa m'malo mwa dTTP mu machitidwe a PCR ndipo chinthu chokulitsa cha PCR chomwe chili ndi maziko a dU chimapangidwa, puloteniyo imatha kuphwanya glycosidic chomangira cha U maziko muzitsulo imodzi ndi ziwiri. DNA ndikunyozetsa chinthu chokulitsa cha PCR.
Ntchito yovomerezeka
Kukulitsa Kuteteza Kuipitsidwa
Mkhalidwe Wosungira
-20 ° C posungira nthawi yayitali, ziyenera kusakanizidwa bwino musanagwiritse ntchito, pewani kuzizira pafupipafupi.
Bafa yosungirako
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Tanthauzo la Chigawo
Kuchuluka kwa enzyme yofunikira kuti iwononge 1µg ya DNA ya chingwe chimodzi chokhala ndi maziko a dU mu ola la 1 pa 37 ° C ndi 1 unit.
Kuwongolera Kwabwino
1.SDS-PAGE electrophoretic chiyero choposa 98%
2.Kukhudzika kukhudzika, kuwongolera kwa batch-to-batch, kukhazikika
3.Pambuyo pa 1U ya UNG yathandizidwa pa 50 ℃ kwa 2mins, template yomwe ili ndi U pansipa 103 makope iyenera kunyonyotsoka kwathunthu ndipo palibe chokulitsa chomwe chingapangidwe.
4.Palibe exogenous nuclease ntchito
Malangizo
| Zigawo | Kuchuluka (μL) | Kukhazikika komaliza |
| 10 × PCR Buffer (dNTP yaulere, Mg²+mfulu) | 5 | 1 × pa |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (m'malo mwa 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 × Primer Mixa | 2 | 1 × pa |
| Template | - | ＜1μg/machitidwe |
| ddH₂O | ku 50 | - |
Zindikirani: a: Ngati ntchito qPCR/qRT-PCR, kafukufuku fulorosenti ayenera kuwonjezeredwa mu anachita dongosolo.Kawirikawiri, chigawo chomaliza cha 0.2 μM chingapereke zotsatira zabwino;pamene machitidwe sakuyenda bwino, ndende yoyambira imatha kusinthidwa mumitundu ya 0.2-1 μM.Nthawi zambiri, ndende ya kafukufukuyo imakonzedwa bwino mumitundu ya 0.1-0.3 μM.Kuyesa kwa concentration gradient kumatha kuchitidwa kuti mupeze kuphatikiza kwabwino kwambiri kwa primer ndi probe.
Zolemba
1.UNG enzyme ingagwiritsidwe ntchito kuchotsa zinthu zokulitsa za dUTP zomwe zaipitsidwa kuchokera pamachitidwe a PCR amplification reaction isanachitike, ndikupewa zotsatira zabodza chifukwa cha kuipitsidwa kwazinthu.
2.Kutentha koyenera kwa UNG enzyme kuti igwiritsidwe ntchito polimbana ndi kuipitsidwa kwa PCR nthawi zambiri ndi 50 ℃ kwa 2mins;kusatsegula ndi 95 ℃ kwa 5mins.
3.Pewani kuzizira pafupipafupi, ndipo musamawonetse kutentha kwakukulu.
4.Mitundu yosiyanasiyana yokulitsidwa imakhala ndi magwiridwe antchito osiyanasiyana a dUTP komanso kukhudzidwa kwa enzyme ya UNG, chifukwa chake, ngati kugwiritsa ntchito kachitidwe ka UNG kumabweretsa kuchepa kwa kuzindikira, kachitidwe kachitidwe kamayenera kusinthidwa ndikukometsedwa, ngati mukufuna thandizo laukadaulo, chonde lemberani. kampani yathu.
