UDG/UNG Enzymes
UDG(uracil DNA glycosylase) e mafai ona fa'aosoina le fa'asusu o le N-glycosidic so'otaga i le va o le uracil base ma le suka-phosphate ivi i le ssDNA ma le dsDNA.E mafai ona faigofie ona pulea le filogia aerosol ma e talafeagai mo faiga masani mole biology e pei ole PCR, qPCR, RT-qPCR ma LAMP.
Fa'amatalaga
| Fa'aaliga Host | Recombinant E. coliwith uracil DNA glycosylase gene |
| Molecular Weight | 24. 8kDa |
| Mama | ≥95% (SDS-PAGE) |
| Fa'aleaogaina o le vevela | 95℃, 5~10 min |
| Fa'amatalaga Vaega | E tasi le iunite (U) o loʻo faʻamatalaina o le aofaʻi o le enzyme e manaʻomia e faʻapipiʻi ai le hydrolysis o le l μg dU o loʻo iai le dsDNA i le 30 minute i le 25 ℃. |
Teuina
E tatau ona teuina le oloa i le-25 ℃ ~ -15 ° C mo le lua tausaga.
Faatonuga
1.Sauniuniga o le paluga tali PCR e tusa ai ma le faiga o lo'o mulimuli mai
| Vaega | Volume (μL) | Fa'atonuga mulimuli |
| 10×PCR Puipui (Mg²+Plus) | 5 | 1× |
| 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 mamanu | X | - |
| Muamua1 (10μmol/L) | 2 | 0.4 μmol/L |
| Muamua 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 | E oo atu i le 50 | - |
Fa'aaliga: E tusa ai ma faʻataʻitaʻiga manaʻoga, o le faʻamaualuga mulimuli o le dUTP e mafai ona fetuʻunaʻi i le va o le 0.2-0.6 mmol / L, ma le 0.2 mmol / L dTTP e mafai ona faʻaopoopoina filifilia.
2.Fa'aliga fa'aopoopo
| Laasaga taamilo | vevela | Taimi | Ta'amilosaga |
| fa'aleaga fa'ata'ita'iga o lo'o iai le dU | 25℃ | 10min | 1 |
| UDG fa'agaoioia, fa'ata'ita'iga muamua denaturation | 95℃ | 5~10min | 1 |
| Denaturation | 95℃ | 10 sec |
30-35 |
| Fa'asusu | 60℃ | 20 sec | |
| Fa'aopoopoga | 72 ℃ | 30sec/kb | |
| Fa'aopoopoga mulimuli | 72 ℃ | 5 minu | 1 |
Fa'aaliga: Ole taimi ole tali ile 25°C e mafai ona fetuunai ile 5-10mins e tusa ai ma manaoga faʻataʻitaʻi.
Fa'amatalaga
1.UDG o loʻo galue i le tele o PCR tali faʻafefe.
2.Enzymes e tatau ona teuina i totonu o se pusa aisa poʻo luga ole taele aisa pe a faʻaaogaina, ma e tatau ona teuina ile-20°C i le taimi lava e uma ai le faʻaaogaina.
3.Fa'amolemole fai le PPE e mana'omia, o se ofu su'esu'e ma totigilima, ia mautinoa lou soifua maloloina ma lou saogalemu!
