UDG/UNG Ii-Enzymes
I-UDG (uracil DNA glycosylase) inokwenza i-hydrolysis ye-N-glycosidic ikhonkco phakathi kwesiseko se-uracil kunye ne-sugar-phosphate backbone kwi-ssDNA kunye ne-dsDNA.Iyakwazi ukulawula lula ungcoliseko lwe-aerosol kwaye ifanelekile kwiinkqubo zebhayoloji eziqhelekileyo ze-molecular ezifana ne-PCR, qPCR, RT-qPCR kunye ne-LAMP.
Iinkcukacha
| Isivakalisi sokuvakalisa | Recombinant E. coliwith uracil DNA glycosylase gene |
| Ubunzima beMolekyuli | 24. 8kDa |
| Ubunyulu | ≥95% (SDS-PAGE) |
| Ukushisa Ukungasebenzi | 95℃, 5 ~ 10 imiz |
| Inkcazelo yeyunithi | Iyunithi enye (U) ichazwa njengobungakanani be-enzyme efunekayo ukuze i-catalyze i-hydrolysis ye-l μg dU equlethe i-dsDNA kwimizuzu engama-30 kwi-25℃. |
Ugcino
Imveliso kufuneka igcinwe kwi-25℃~-15°C iminyaka emibini.
Imiyalelo
1.Ukulungiswa komxube wokusabela kwe-PCR ngokwenkqubo elandelayo
| Amacandelo | Umthamo (μL) | Ugxininiso lokugqibela |
| 10×PCR Buffer (Mg²+Plus) | 5 | 1× |
| 25 mmol/LMgCl | 3 | 1.5 mmol/L |
| idUTP(10 mmol/L) | 3 | 0.6 mmol/L |
| I-dCTP/dGTP/dATP/dTTP(10mmol/Leach) | 1 | 0.2 mmol / Leach |
| Isakhelo seDNA | X | - |
| I-Primer1 (10μmol/L) | 2 | 0.4 μmol/L |
| I-Primer 2 (10μmol/L) | 2 | 0.4 μmol/L |
| I-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 | Ukuya kuthi ga kwi-50 | - |
Phawula: Ngokweemfuno zovavanyo, ukugxininiswa kokugqibela kwe-dUTP kunokulungelelaniswa phakathi kwe-0.2-0.6 mmol / L, kunye ne-0.2 mmol / L dTTP inokongezwa ngokukhethiweyo.
2.Inkqubo yokwandisa
| Inyathelo lomjikelo | Ubushushu | Ixesha | Imijikelo |
| I-dU equlathe ukuthotywa kwetemplate | 25℃ | 10mins | 1 |
| Ukuqaliswa kwe-UDG, i-template yokuqala denaturation | 95℃ | 5 ~ 10mins | 1 |
| I-Denaturation | 95℃ | 10 imizuzwana |
30-35 |
| Ukuhlaziya | 60℃ | 20 imizuzwana | |
| Ulwandiso | 72℃ | 30sec/kb | |
| Ukongezwa kokugqibela | 72℃ | 5 imiz | 1 |
Phawula: Ixesha lokuphendula kwi-25 ° C linokulungiswa ngaphakathi kwe-5-10mins ngokweemfuno zokulinga.
Amanqaku
1.I-UDG iyasebenza kwi-PCR reaction buffers ezininzi.
2.Ii-enzyme kufuneka zigcinwe kwi-icebox okanye kwindawo yokuhlamba umkhenkce xa isetyenziswa, kwaye kufuneka igcinwe kwi-20 ° C ngokukhawuleza emva kokusetyenziswa.
3.Nceda unxibe iPPE eyimfuneko, idyasi yelebhu kunye neeglavu, ukuqinisekisa impilo nokhuseleko lwakho!
