Ma enzymes a UDG/UNG
UDG(uracil DNA glycosylase) imatha kuyambitsa hydrolysis ya ulalo wa N-glycosidic pakati pa uracil base ndi msana wa shuga-phosphate mu ssDNA ndi dsDNA.Imatha kuwongolera kuipitsidwa kwa aerosol mosavuta ndipo ndi yoyenera pamakina wamba a biology monga PCR, qPCR, RT-qPCR ndi LAMP.
Zofotokozera
| Expression Host | Recombinant E. coliwith uracil DNA glycosylase jini |
| Kulemera kwa Maselo | 24.8k ndi |
| Chiyero | ≥95% (SDS-PAGE) |
| Kutentha Kutentha | 95 ℃, 5 ~ 10 min |
| Tanthauzo la Chigawo | Chigawo chimodzi (U) chimatanthauzidwa ngati kuchuluka kwa enzyme yomwe imafunikira kuti ipangitse hydrolysis ya l μg dU-containing dsDNA mu mphindi 30 pa 25 ℃. |
Kusungirako
Zogulitsa ziyenera kusungidwa pa -25 ℃ ~ -15 ° C kwa zaka ziwiri.
Malangizo
1.Kukonzekera PCR anachita osakaniza motsatira dongosolo
| Zigawo | Kuchuluka (μL) | Kukhazikika komaliza |
| 10×PCR Buffer (Mg²+Plus) | 5 | 1 × pa |
| 25 mmol/LMgCl | 3 | 1.5 mmol / L |
| dUTP (10 mmol/L) | 3 | 0.6 mmol / L |
| dCTP/dGTP/dATP/dTTP(10mmol/Leach) | 1 | 0.2 mmol / Leach |
| DNA template | X | - |
| Choyamba1 (10μmol/L) | 2 | 0.4 μmol/L |
| Woyamba 2 (10μmol/L) | 2 | 0.4 μmol/L |
| Taq DNA Polymerase (5 U/μL) | 0. 5 | 0.05 U/μL |
| Uracil DNA Glycosylase (UDG/UNG), 1 U/μL | 1 | 1 U/μL |
| ddH₂O | Mpaka 50 | - |
Zindikirani: Malingana ndi zofunikira zoyesera, chiwerengero chomaliza cha dUTP chikhoza kusinthidwa pakati pa 0.2-0.6 mmol / L, ndi 0.2 mmol / L dTTP akhoza kuwonjezeredwa mwa kusankha.
2.Ndondomeko ya amplification
| Sitepe yozungulira | Kutentha | Nthawi | Zozungulira |
| Kuwonongeka kwa template yokhala ndi dU | 25 ℃ | 10 mins | 1 |
| UDG activation, template yoyamba denaturation | 95 ℃ | 5-10 mphindi | 1 |
| Denaturation | 95 ℃ | 10 sec |
30-35 |
| Annealing | 60 ℃ | 20 sec | |
| Kuwonjezera | 72 ℃ | 30sec/kb | |
| Kuwonjezera komaliza | 72 ℃ | 5 min | 1 |
Zindikirani: Nthawi yochitira pa 25 ° C ikhoza kusinthidwa mkati mwa 5-10mins malinga ndi zofunikira zoyesera.
Zolemba
1.UDG imagwira ntchito m'mabufa ambiri a PCR.
2.Ma enzyme amayenera kusungidwa mu ayezi kapena pamadzi osambira akagwiritsidwa ntchito, ndipo ayenera kusungidwa pa -20 ° C atangogwiritsa ntchito.
3.Chonde valani PPE yofunikira, malaya a labu ndi magolovesi, kuti mutsimikizire thanzi lanu ndi chitetezo!
