Kutentha Kwambiri UNG
Temperature Sensitive UNG (TS-UNG) ndi yopezedwa ndi mawu ophatikizanso mu E. coli.Enzymeyi imathandizira kutulutsidwa kwa uracil waulere kuchokera ku DNA yokhala ndi uracil imodzi ndi mizere iwiri ndipo sigwira ntchito motsutsana ndi RNA.Poyerekeza ndi wamba UNG puloteni wa E. coli jini chiyambi, TS-UNG puloteni ali ndi ntchito apamwamba pa kutentha otsika (20 ℃ ~ 37 ℃) ndi kutentha kutentha ndi inactivated mosavuta (50 ℃), kupewa kuwonongeka kwa dUTP munali matalikidwe. zopangidwa ndi kutentha kwa firiji ndi zochitika zotsalira zomwe zingakhalepo pambuyo poyambitsa enzyme ya UNG.Chifukwa chake, enzyme ya TS-UNG siyoyenera kutengera kupewa kuipitsidwa kwa PCR, komanso imagwirizana bwino ndi pulogalamu yokulitsa ya RT-PCR ndipo ingagwiritsidwe ntchito popewa kuipitsidwa kwa RT-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 7.5) , 100 mM NaCl, 0.1 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 limodzi pa 37 ° C ndi 1 unit of activity (U).
Kuwongolera Kwabwino
1.SDS-PAGE electrophoretic chiyero choposa 98%
2.Ntchito yowonongeka, kulamulira kwa batch-to-batch, kukhazikika
3.Palibe exogenous nuclease ntchito, palibe exogenous endonuclease kapena exonuclease kuipitsidwa.
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 |
| 1 U/μLTS-UNG | 0.5 (0.1-0.5) | 0.5 U (0.1-0.5U) |
| 25 × Primer Mixa | 2 | 1 × pa |
| Template | - | ＜1μg/machitidwe |
| ddH₂O | ku 50 | - |
Zindikirani: a: Ngati ntchito qPCR/qRT-PCR, kafukufuku fulorosenti ayenera kuwonjezeredwa mu kachitidwe kachitidwe.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.Kutentha koyenera kwa TS-UNG enzyme ndikochepa kwambiri, ndipo kumatha kukhathamiritsa mumitundu ya 20 ℃ ~ 37 ℃, mlingo wa enzyme ndi nthawi yochitira ukhoza kukongoletsedwa mumitundu ya 0.1 ~ 0.5 U, 5 ~ 10 min;ndipo puloteniyo imatha kuyimitsidwa polemba m'mbuyo.
2.Ndi yoyenera kwa PCR ndi RT-PCR kuteteza kuipitsidwa.
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.
