I-Superstart qPCR Premix plus-UNG
Inombolo yekati: HCB5071E
I-Superstart qPCR Premix plus-UNG iyi-reagent ekhethekile eyenzelwe i-Real Time PCR ukusabela kwekhwalithi nenani kusetshenziswa ukutholwa okusekelwe ku-probe, okuthuthukiswe ngokukhethekile izinqubo ze-lyophilization.Iqukethe i-enzyme ye-hotstart Taq plus (DG) enoveli, enomsebenzi wayo we-enzyme ye-Taq evalwe ekamelweni lokushisa, ivimbela ngempumelelo ukukhuliswa okungaqondile okubangelwa i-primer non-specific annealing noma ukwakheka kwe-primer dimer ngaphansi kwezimo eziphansi zokushisa, ngaleyo ndlela kube ngcono. ukucaciswa kokusabela kwe-amplification.Lesi sikhungo sisebenzisa ibhafa ethile ye-qPCR elungiselelwe kanye nesistimu yokunqanda ukungcoliswa kwe-UNG/dUTP ukuze kuzuzwe ukuqala okushisayo, ukuthuthukisa kakhulu ukusebenza kahle nokuzwela kokusabela kwe-qPCR.Ingathola amajika amahle ajwayelekile ezindaweni eziningi zokulinganisa futhi yenze ukulinganisa ngokunembile, ivimbele ngempumelelo ukukhuliswa okungamanga okubangelwa imikhiqizo eyinsalela ye-PCR noma ukungcoliswa kwe-aerosol.Lesi sikhungo sihambisana nezinsimbi eziningi ze-PCR zomthamo we-fluorescent ezivela kubakhiqizi abafana ne-Applied Biosystems, i-Eppendorf, i-Bio- Rad ne-Roche njll., futhi ibonisa ukuzinza okuhle ngendlela e-lyophilized.
Ukwakhiwa kwe-reagent
1. 5×HotstartPremix plus-UNG (Mg2+mahhala) (DG)
2. 250 mM MgCl2
3. 4× lyoprotectant (kuyakhetheka)
Izimo Zokugcina
Ukugcina isikhathi eside -20 ℃;ingagcinwa ku-4℃ kuze kube yizinyanga ezi-3.Hlanganisa kahle ngaphambi kokusetshenziswa futhigwema ukuqhwaza okuphindaphindiwe nokuncibilika.
I-Cycling Protocol
| Inqubo | Temp. | Isikhathi | Umjikelezo |
| Ukugaya ukudla | 50℃ | 2 imiz | 1 |
| Ukusebenza kwe-Polymerase | 95℃ | 1~5 iminithi | 1 |
| I-Denature | 95℃ | 10~20 imizuzwana | 40-50 |
| I-Anealing kanye Nokwengezwa | 56 ~ 64℃ | 20~60 imizuzwana | 40-50 |
I-qPCR Liquid Reaction Syisiqu Ukulungiselela
|
Ukwakheka |
25µL ivolumu |
50µL Ivolumu |
Ukugxila Kokugcina |
| 5×HotstartPremix plus-UNG(Mg2+mahhala) (DG) | 5µL | 10µL | 1× |
| 250mM MgCl2 | 0.45µL | 0.9µL | 4.5 mM |
| I-4 × i-lyoprotectant1 | 6.25µL | 12.5µL | 1× |
| 25×Primer-Probe Mix2 | 1µL | 2µL | 1× |
| I-DNA yesifanekiso3 | —- | —- | —- |
| ddH2O | Ku-25µL | Ku-50µL | —- |
1. Ukugxila kokugcina kwe-0.2μM kuma-primers ngokuvamile kuletha imiphumela emihle;lapho ukusebenza kokusabela kukubi, lungisa ukugxilisa kwe-primer phakathi kwebanga lika-0.2-1μM njengoba kudingeka.Ukugxiliswa kwe-probe ngokuvamile kulungiselelwa phakathi kwebanga elingu-0.1-0.3μM ngokuhlolwa kwegradient ukuze kutholwe izinhlanganisela ezifanele.
2. Inombolo yekhophi yezakhi zofuzo eziqondiwe eziqukethwe ezinhlotsheni ezihlukene zezifanekiso ziyahlukahluka;uma kunesidingo, ukuhlanjululwa kwe-gradient kungenziwa ukuze kunqunywe inani elifanele lesengezo sesifanekiso.
3. Lesi simiso singaba lyophilized;lapho amakhasimende esebenzisa lesi simiso ngaphandle kwezidingo zokomisa okokuqandisa, i-4×lyoprotectant ingenziwa ngokukhetha; uma kunemikhiqizo efriziwe edingekayo, ngesikhathi sokuqinisekiswa kokusebenza komkhiqizo we-liquid reagents, kufanele ingeze i-4×lyoprotectant ukuqinisekisa ukuhambisana nezingxenye zesistimu ye-lyophilized. kanye nemiphumela.
Lapho uhlelo lusetshenziswad ukuze umiswe yiqhwa, lungiselela uhlelo as okulandelayo:
| Ukwakheka | 25µL Isistimu yokusabela |
| 5 ×HotstartPremix plus-UNG (Mg2+mahhala) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| I-4 × i-lyoprotectant | 6.25µL |
| 25×Primer-Probe Mix | 1µL |
| ddH2O | Ku-18 ~ 20µL |
* Uma kudingeka ezinye izinhlelo zokumisa iqhwa, sicela uthintane ngokuhlukene.
Inqubo ye-Lyophilizationss
| Inqubo | Temp. | Isikhathi | Isimo | Ingcindezi |
| Ukubanda kwangaphambili | 4℃ | 30 imiz | Bamba |
1 atm |
| -50 ℃ | 60 imiz | Ukupholisa | ||
| -50 ℃ | 180 imiz | Bamba | ||
| Ukomisa Okuyisisekelo | -30 ℃ | 60 imiz | Ukushisisa |
I-Ultimate Vacuum |
| -30 ℃ | 70 imiz | Bamba | ||
| Ukomisa okwesibili | 25℃ | 60 imiz | Ukushisisa |
I-Ultimate Vacuum |
| 25℃ | 300 min | Bamba |
2. Inqubo ye-lyophilization engenhla ingeyereferensi kuphela.Izinhlobo ezihlukene zemikhiqizo kanye nezindawo zokomisa iqhwa ezahlukene zinemingcele ehlukene, ngakho-ke ukulungiswa kungenziwa ngokuya ngokwangempela.izimo ngesikhathi sokusetshenziswa.
3. Izinqubo ezihlukene ze-lyophilization zingase zifaneleke osayizi abahlukene beqoqo be-lyophilizedimikhiqizo, ngakho-ke ukuqinisekiswa kokuhlola okwanele kufanele kwenziwe lapho kusetshenziselwa ukukhiqizwa okukhulu.
Imiyalo yokusebenzisa i-lyophilized powder
1. Kafushane centrifuge impushana lyophilized;
2. Faka ithempulethi ye-nucleic acid kumpuphu e-lyophilized bese wengeza amanzi afika ku-25µL;
3. Hlanganisa kahle nge-centrifugation futhi usebenzise umshini.
Ikhwalithi yokulawula:
1. Ukuhlola okusebenzayo: ukuzwela, ukucaciswa, ukukhiqizwa kabusha kwe-qPCR.
2. Akukho msebenzi we-nuclease exogenous, akukho endogenous endo/exonuclease ukungcola.
Ulwazi Lokusebenza:
1. I-Superstart qPCR Premix plus-UNG isebenzisa i-enzayimu inoveli eshisa izikhotha evumela ukuqalisa ukushisa ngokushesha phakathi kwemizuzu engu-1 ~ 5;ngokusebenzisa ukwakheka okukhethekile kwebhafa ifanele ukusabela komthamo we-PCR we-multiplex fluorescent.
2. Inokucaciswa okuphezulu okuthuthukisa kakhulu ukuzwela kokutholwa komkhawulo we-PCR we-fluorescence quantitative, okwenza amajika okukhulisa ukuphakama abejwayelekile, inani le-fluorescence lithole ukuthuthukiswa okusobala kuzifanekiso zokugxilisa eziphansi, ezifanele njengama-reagents e-PCR azwela kakhulu e-fluorescence.
3. Kuma-primer anezinga lokushisa eliphansi le-annealing noma ama-fragment amade kuno-200bp, indlela yezinyathelo ezi-3 iyanconywa.
4. Ukusebenza kahle kokusetshenziswa kwe-dUTP kanye nokuzwela i-enzyme ye-UNG kuyahluka kuzakhi zofuzo eziqondiwe ezihlukene, ngakho-ke uma ukusebenzisa uhlelo lwe-UNG kuholela ekwehleni kokuzwela kokutholwa, isistimu yokusabela kufanele ilungiswe futhi yenziwe kahle.Uma ukusekelwa kwezobuchwepheshe kuyadingeka sicela uxhumane nenkampani yethu.
5. Sebenzisa izindawo ezizinikele kanye namapayipi ngaphambi nangemva kokukhulisa, gqoka amagilavu ngesikhathi sokusebenza futhi uwashintshe njalo;ungavuli ishubhu lokusabela ngemva kokuqedwa kwe-PCR ukuze unciphise ukungcoliswa kwamasampula ngemikhiqizo ye-PCR.
