Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG kapa UDG) ke motsoako oa motsoako oa E.coli o nang le boima ba molek'hule oa 25 kDa.E etsa hore ho lokolloe uracil e sa lefelloeng ho tsoa ho DNA e nang le mela e le 'ngoe le e nang le likarolo tse peli,' me ha e sebetse khahlanong le RNA, 'me e ka sebelisoa ho thibela tšilafalo ea lihlahisoa tsa amplification tsa PCR.Molao-motheo oa ts'ebetso o ipapisitse le 'nete ea hore haeba dUTP e nkeloa sebaka ke dTTP karabelong ea PCR mme sehlahisoa sa amplification sa PCR se nang le metheo ea dU se thehiloe, enzyme e ka khetha ka mokhoa o ikhethileng tlamahano ea glycosidic ea metheo ea U ka likhoele tse le 'ngoe le tse habeli. DNA le ho nyenyefatsa sehlahisoa sa amplification sa PCR.
E khothalelitsoeng Kopo
Matlafatso ea Thibelo ea Tšilafalo
Boemo ba polokelo
-20°C bakeng sa polokelo ea nako e telele, e lokela ho tsoakoa hantle pele e sebelisoa, qoba ho qhibiliha khafetsa.
Sebaka sa polokelo
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Tlhaloso ea Yuniti
Palo ea enzyme e hlokahalang ho theola 1µg ea DNA e nang le khoele e le 'ngoe e nang le li-bases tsa dU ka hora e le 1 ho 37°C ke yuniti e le' ngoe.
Taolo ea Boleng
1.SDS-PAGE ho hloeka ha electrophoretic ho feta 98%
2.Amplification sensitivity, taolo ea batch-to-batch, botsitso
3.Kamora hore 1U ea UNG e tšoaroe ho 50 ℃ bakeng sa 2mins, template e nang le U ka tlase ho likopi tse 103 e lokela ho senyeha ka botlalo mme ha ho sehlahisoa sa amplification se ka hlahisoang.
4.Ha ho ts'ebetso ea exogenous nuclease
Litaelo
| Likaroloana | Bolumo (μL) | Khatiso ea ho qetela |
| 10 × PCR Buffer (dNTP mahala, Mg²+mahala) | 5 | 1× |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (emisa dTTP) | - | 200-600 μM |
| 25 limilimithara MgCl2 | 2-8 μL | 1-4 mmM |
| 5 U/μL Taq | 0.25 | 1.25 U |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 25 × Primer Mixa | 2 | 1× |
| Setšoantšo | - | ＜1μg/karabelo |
| ddH₂O | Ho ea ho 50 | - |
Hlokomela: a: Haeba e sebelisoa bakeng sa qPCR/qRT-PCR, probe ea fluorescent e lokela ho kenngoa tsamaisong ea karabelo.Hangata, palo ea ho qetela ea 0,2 μM e ka fana ka liphello tse ntle;ha ts'ebetso ea karabelo e fokola, mahloriso a primer a ka fetoloa maemong a 0.2-1 μM.Hangata, mahloriso a probe a ntlafatsoa maemong a 0.1-0.3 μM.Liteko tsa Concentration gradient li ka etsoa ho fumana motsoako o motle ka ho fetisisa oa primer le probe.
Lintlha
1.Enzyme ea UNG e ka sebelisoa ho tlosa lihlahisoa tsa amplification tsa dUTP tse silafetseng tsamaisong ea karabelo pele ho karabelo ea amplification ea PCR, ebe ho qoba liphetho tse fosahetseng ka lebaka la tšilafalo ea sehlahisoa.
2.Thempereichara e nepahetseng bakeng sa enzyme ea UNG e tla sebelisoa karabelong ea PCR e khahlanong le tšilafalo hangata ke 50 ℃ bakeng sa 2mins;boemo ba ho se sebetse ke 95 ℃ bakeng sa 5mins.
3.Qoba ho qhibiliha khafetsa, 'me u se ke ua pepesehela ho feto-fetoha ho hoholo ha mocheso.
4.Liphatsa tsa lefutso tse fapaneng tse lokelang ho holisoa li na le ts'ebeliso e fapaneng ea ts'ebeliso ea dUTP le kutloelo-bohloko ho enzyme ea UNG, ka hona, haeba ts'ebeliso ea sistimi ea UNG e lebisa ho fokotseheng ha kutlo, sistimi ea karabelo e lokela ho lokisoa le ho ntlafatsoa, haeba o hloka tšehetso ea tekheniki, ka kopo ikopanye. khampani ea rona.
