Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG ko UDG) wani recombinant clone ne na E.coli tare da nauyin kwayoyin halitta na 25 kDa.Yana haifar da sakin uracil na kyauta daga uracil mai ƙunshe da DNA guda ɗaya da madauri biyu, kuma baya aiki da RNA, kuma ana iya amfani dashi don hana gurɓata samfuran haɓaka PCR.Ka'idar aiki ta dogara ne akan gaskiyar cewa idan an maye gurbin dUTP don dTTP a cikin amsawar PCR kuma an samar da samfurin haɓakawa na PCR wanda ke ɗauke da sansanonin dU, enzyme na iya zaɓin karya haɗin glycosidic na tushen U a cikin ɗaki ɗaya da mai ɗaure biyu. DNA da lalata samfurin haɓakawa na PCR.
Aikace-aikacen da aka ba da shawarar
Ƙarfafa Rigakafin Ƙira
Yanayin Ajiya
-20 ° C don ajiya na dogon lokaci, ya kamata a gauraye da kyau kafin amfani, kauce wa daskare-narke akai-akai.
Ma'ajiyar ajiya
20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Ma'anar Naúrar
Adadin enzyme da ake buƙata don ƙasƙantar da 1µg na DNA mai ɗauri ɗaya mai ɗauke da sansanonin dU a cikin awa 1 a 37°C shine raka'a 1.
Kula da inganci
1.SDS-PAGE tsarkin electrophoretic fiye da 98%
2.Amplification hankali, sarrafa tsari-zuwa-tsari, kwanciyar hankali
3.Bayan an yi maganin 1U na UNG a 50 ℃ na 2mins, samfurin da ke ɗauke da U da ke ƙasa da kwafi 103 ya kamata a lalata su gaba ɗaya kuma ba za a iya samar da samfurin ƙarawa ba.
4.Babu exogenous nuclease aiki
Umarni
| Abubuwan da aka gyara | girma (μL) | Natsuwa na ƙarshe |
| 10 × PCR Buffer (dNTP kyauta, Mg²+kyauta) | 5 | 1× |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (maye gurbin dTTP) | - | 200-600 μM |
| 25 mMgCl2 | 2-8 ml | 1-4 mm |
| 5 U/μL Taq | 0.25 | 1.25 ku |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 25 × Farko Mixa | 2 | 1× |
| Samfura | - | 1 μg / amsawa |
| ddH₂O | Zuwa 50 | - |
Lura: a: Idan an yi amfani da shi don qPCR/qRT-PCR, ya kamata a ƙara bincike mai kyalli a cikin tsarin amsawa.Yawancin lokaci, ƙaddamarwa na ƙarshe na 0.2 μM na iya ba da sakamako mai kyau;lokacin da aikin amsawa ya kasance mara kyau, za'a iya daidaita ƙaddamar da ƙaddamarwa a cikin kewayon 0.2-1 μM.Yawancin lokaci, an inganta ƙaddamar da bincike a cikin kewayon 0.1-0.3 μM.Za a iya yin gwaje-gwajen hankali don nemo mafi kyawun haɗe-haɗe na firamare da bincike.
Bayanan kula
1.Ana iya amfani da UNG enzyme don cire gurɓataccen samfuran ƙarawa na dUTP daga tsarin amsawa kafin amsawar haɓakawa na PCR, sannan don guje wa sakamako mai kyau na ƙarya saboda gurɓataccen samfur.
2.Mafi kyawun zafin jiki don UNG enzyme da za a yi amfani da shi a cikin maganin cutar PCR gabaɗaya shine 50 ℃ na 2mins;yanayin rashin kunnawa shine 95 ℃ na 5mins.
3.Guji daskare-narke akai-akai, kuma kar a fallasa ga manyan canjin yanayin zafi.
4.Dabbobi daban-daban da za a haɓaka suna da tasirin amfani daban-daban na dUTP da hankali ga UNG enzyme, sabili da haka, idan amfani da tsarin UNG ya haifar da raguwar ganewar ganewa, ya kamata a daidaita tsarin amsawa da ingantawa, idan kuna buƙatar goyon bayan fasaha, tuntuɓi. kamfaninmu.
