Awọn enzymu UDG/UNG
UDG (uracil DNA glycosylase) le ṣe itọsi hydrolysis ti ọna asopọ N-glycosidic laarin ipilẹ uracil ati ẹhin suga-phosphate ni ssDNA ati dsDNA.O le ni rọọrun ṣakoso idoti aerosol ati pe o dara fun awọn ọna ṣiṣe isedale molikula ti o wọpọ gẹgẹbi PCR, qPCR, RT-qPCR ati LAMP.
Awọn pato
| Gbalejo ikosile | Recombinant E. coliwith uracil DNA glycosylase gene |
| Òṣuwọn Molikula | 24.8kDa |
| Mimo | ≥95% (SDS-iwe) |
| Ooru Inactivation | 95℃, 5 ~ 10 iṣẹju |
| Unit Definition | Ẹyọ kan (U) jẹ asọye bi iye henensiamu ti o nilo lati ṣaṣeyọri hydrolysis ti l μg dU ti o ni dsDNA ninu awọn iṣẹju 30 ni 25℃. |
Ibi ipamọ
Ọja naa yẹ ki o wa ni ipamọ ni 25 ℃ ~ -15 ° C fun ọdun meji.
Awọn ilana
1.Igbaradi ti idapọ ifaseyin PCR ni ibamu si eto atẹle
| Awọn eroja | Iwọn didun (μL) | Ifojusi ikẹhin |
| 10×PCR Ifipamọ (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 awoṣe | X | - |
| Alakoko 1 (10μmol/L) | 2 | 0.4 μmol/L |
| Alakoko 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 | Titi di 50 | - |
Akiyesi: Gẹgẹbi awọn ibeere idanwo, ifọkansi ikẹhin ti dUTP le ṣe atunṣe laarin 0.2-0.6 mmol/L, ati 0.2 mmol/L dTTP le ṣe afikun ni yiyan.
2.Ilana imudara
| Igbesẹ iyipo | Iwọn otutu | Aago | Awọn iyipo |
| dU-ti o ni awọn ibaje awoṣe | 25 ℃ | 10 min | 1 |
| Muu ṣiṣẹ UDG, denaturation ibẹrẹ awoṣe | 95℃ | 5-10 iṣẹju | 1 |
| Denaturation | 95℃ | 10 iṣẹju-aaya |
30-35 |
| Annealing | 60℃ | 20 iṣẹju-aaya | |
| Itẹsiwaju | 72℃ | 30 iṣẹju-aaya/kb | |
| Ipari ipari | 72℃ | 5 min | 1 |
Akiyesi: Akoko ifaseyin ni 25 ° C le ṣe atunṣe laarin awọn iṣẹju 5-10 ni ibamu si awọn ibeere idanwo.
Awọn akọsilẹ
1.UDG n ṣiṣẹ ni ọpọlọpọ awọn buffer ifa PCR.
2.Awọn enzymu yẹ ki o wa ni ipamọ sinu apoti yinyin tabi lori yinyin nigba lilo, ati pe o yẹ ki o wa ni ipamọ ni-20 ° C lẹsẹkẹsẹ lẹhin lilo.
3.Jọwọ wọ PPE pataki, iru ẹwu lab ati awọn ibọwọ, lati rii daju ilera ati ailewu rẹ!
