Mocheso o Utloahala UNG
Temperature Sensitive UNG (TS-UNG) e fumanoa ka polelo e kopanyang ho E. coli.Enzyme e hlohlelletsa ho lokolloa ha uracil ea mahala ho DNA e nang le uracil e nang le mela e le 'ngoe le e habeli 'me ha e sebetse khahlano le RNA.Ha ho bapisoa le enzyme e tloaelehileng ea UNG ea tšimoloho ea liphatsa tsa lefutso tsa E. coli, enzyme ea TS-UNG e na le ts'ebetso e phahameng ka mocheso o tlase (20℃~37℃) 'me ha e na mocheso ebile ha e sebetse habonolo (50℃), e qoba ho senyeha ha amplification e nang le dUTP. lihlahisoa ka mocheso oa kamore ka ts'ebetso e setseng e ka setseng kamora ho koaloa ha enzyme e tloaelehileng ea UNG.Ka hona, enzyme ea TS-UNG ha e loketse feela karabelo ea thibelo ea tšilafalo ea PCR, empa hape e ts'oana hantle le lenaneo la amplification la RT-PCR mme e ka sebelisoa ho karabelo ea thibelo ea tšilafalo ea RT-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 7.5) , 100 mM NaCl, 0.1 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 1 unit of activity (U).
Taolo ea Boleng
1.SDS-PAGE ho hloeka ha electrophoretic ho feta 98%
2.Ts'ebetso ea ho nyenyefatsa, taolo ea batch-to-batch, botsitso
3.Ha ho ts'ebetso ea nuclease exogenous, ha ho na exogenous endonuclease kapa tšilafalo ea exonuclease.
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 |
| 1 U/μLTS-UNG | 0.5 (0.1-0.5) | 0.5 U (0.1-0.5U) |
| 25 × Primer Mixa | 2 | 1× |
| Setšoantšo | - | ＜1μg/karabelo |
| ddH₂O | Ho ea ho 50 | - |
Tlhokomeliso: a: Haeba e sebelisoa bakeng sa qPCR/qRT-PCR, mochine oa fluorescent o 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.Mocheso o nepahetseng oa karabelo ea enzyme ea TS-UNG e batla e le tlase, 'me e ka ntlafatsoa ho tloha ho 20 ℃ ~ 37 ℃, tekanyo ea enzyme le nako ea karabelo e ka ntlafatsoa ka bongata ba 0.1 ~ 0.5 U, 5 ~ 10 mets;mme enzyme e ka emisoa ts'ebetsong ea ho fetolela ka morao.
2.E loketse PCR le RT-PCR ho thibela tšilafalo.
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.
