UDG/UNG Enzymes
Ka taea e UDG(uracil DNA glycosylase) te whakakorikori i te hauwai o te hononga N-glycosidic i waenga i te turanga uracil me te tuara huka-phosphate i roto i te ssDNA me te dsDNA.Ka taea e ia te whakahaere ngawari i te parahanga aerosol, he pai hoki mo nga punaha koiora ngota noa penei i te PCR, qPCR, RT-qPCR me te LAMP.
Whakatakotoranga
| Kaihautu Whakaaturanga | Recombinant E. coliwith uracil DNA glycosylase ira |
| Taumaha Molecular | 24. 8kDa |
| Maamaa | ≥95% (SDS-WĀRANGI) |
| Te Wera Whakakore | 95℃, 5~10 min |
| Whakamaramatanga Waeine | Kotahi te waeine (U) kua tautuhia ko te nui o te whaariki e hiahiatia ana hei whakakorikori i te hauwai o te l μg dU-kei roto dsDNA i roto i te 30 meneti i te 25 ℃. |
Rokiroki
Me penapena te hua ki-25 ℃ ~ -15 ° C mo nga tau e rua.
Tohutohu
1.Te whakarite i te ranunga tauhohenga PCR kia rite ki te punaha e whai ake nei
| Waehanga | Rōrahi (μL) | Te kukū whakamutunga |
| 10×PCR Pūreirei (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 tauira | X | - |
| Pūmatua1 (10μmol/L) | 2 | 0.4 μmol/L |
| Tuatahi 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 | Tae atu ki te 50 | - |
Tuhipoka: E ai ki nga whakaritenga whakamatautau, ka taea te whakarereke i te kukū whakamutunga o te dUTP i waenga i te 0.2-0.6 mmol/L, me te 0.2 mmol/L dTTP ka taea te taapiri maataki.
2.Te tikanga whakakaha
| Huahi huringa | Te pāmahana | Te wa | Nga huringa |
| te whakahekenga tauira kei roto dU | 25℃ | 10 meneti | 1 |
| Whakahohenga UDG, tauira denaturation tuatahi | 95℃ | 5~10 meneti | 1 |
| Te whakahekenga | 95℃ | 10 hēkona |
30-35 |
| Te whakaene | 60 ℃ | 20 hēkona | |
| Toronga | 72℃ | 30 hēkona/kb | |
| Toronga whakamutunga | 72℃ | 5 min | 1 |
Tuhipoka: Ko te wa urupare i te 25 ° C ka taea te whakatika i roto i te 5-10mins kia rite ki nga whakaritenga whakamatautau.
Notes
1.He kaha te UDG i roto i te nuinga o nga parepare tauhohenga PCR.
2.Me penapena nga enzyme ki roto i te pouaka tio, ki runga ranei i te pati hukapapa ina whakamahia ana, me pupuri ki te-20°C i muri tonu i te whakamahinga.
3.Tena koa mauhia nga PPE e tika ana, he koti taiwhanga me nga karapu, kia pai ai to hauora me to haumaru!
