5×Neoscript Fast RT-qPCR Premix plus-UNG
Inombolo yekati: HCB5142A
I-Neoscript Fast RT Premix-UNG (Probe qRT-PCR) iyingxube ezinzile yeshubhu eyodwa esekelwe kuphenyi elungele ukubhalwa kwesinyathelo esisodwa sokuhlehla kanye ne-PCR yobuningi (qRT-PCR).Isekela ukuxutshwa kwangaphambili kwama-primers nama-probe futhi uhlale uzinzile ngemva kokugcinwa isikhathi eside ezingeni lokushisa eliphansi.Isampula okufanele sihlolwe singengezwa ngokuqondile lapho kusetshenziswa, ngaphandle kokuvula/ukufakwa kwamapayipi okwengeziwe.Lo mkhiqizo uhlinzeka ngezingxenye, isb i-DNA polymerase eqala ukushisa, i-M-MLV, i-labile-labile uracil DNA glycosylase (TS-UNG), i-RNase Inhibitor, i-MgCl2, ama-dNTP (nge-dUTP esikhundleni se-dTTP), neziqinisi.Nge-amplification esheshayo ye-reverse transcriptase elungiswe ngofuzo kanye ne-DNA polymerase, kungenzeka ukuqedela ukukhulisa i-PCR phakathi nemizuzu engama-20-40.Lesi sithako sisebenzisa isigcinalwazi esikhethekile se-qPCR esinama-enzyme axubile e-anti-inhibitory amplification enzyme kanye ne-UNG enzyme.Ngakho-ke, ingathola ukukhuliswa okuhle kwezakhi zofuzo eziqondiwe futhi ivimbele ukukhuliswa okungamanga okubangelwa ukungcoliswa kwensalela ye-PCR kanye ne-aerosol.Lesi sikhungo sihambisana namathuluzi amaningi e-PCR e-fluorescence avela kubakhiqizi abafana ne-Applied Biosystems, i-Eppendorf, i-Bio-Rad ne-Roche.
Isakhi
1.25×Neoscript Fast RTase/UNG Mix
2.I-5×Neoscript Fast RT Premix Buffer (dUTP)
Izimo Zokugcina
Zonke izingxenye kufanele zigcinwe ku -20 ℃ ukuze zigcinwe isikhathi eside futhi 4℃ kuze kube yizinyanga ezi-3.Sicela uhlanganise kahle ngemva kokuncibilika kanye ne-centrifuge ngaphambi kokusebenzisa.Gwema ukuncibilika kweqhwa njalo.
Ukulungiswa Kwesistimu Yokusabela kwe-qRT-PCR
| Izingxenye | 25μLUhlelo | 50μLUhlelo | Ukugxila Kokugcina |
| I-5×Neoscript Fast RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
| 25×Neoscript Fast RTase/UNG Mix | 1μL | 2μL | 1× |
| 25×Primer-Probe Mixa | 1μL | 2μL | 1× |
| I-RNA yesifanekisob | - | - | - |
| ddH2O | Kufika ku-25μL | Kufika ku-50μL | - |
1) a.Ukuhlushwa kokugcina kweprimer kuvame ukuba ngu-0.2μM.Ukuze uthole imiphumela engcono, ukugxiliswa kwe-primer kungenziwa kahle phakathi kwebanga lika-0.2-1μM.Ngokuvamile, ukugxila kwe-probe kungenziwa kahle phakathi kwebanga lika-0.1-0.3μM.
2) b.Lapho usebenzisa Inqubo ye-PCR esheshayo, ukukhulisa ukugxila kweziqalo kanye nama-probe kungase kuphumele emiphumeleni engcono yokukhulisa, futhi isilinganiso sazo kufanele sithuthukiswe ngokufanele.
3) Izinhlobo ezahlukene zamasampuli ziqukethe izinhlobo ezahlukene kanye nokuqukethwe kwe-inhibitor kanye nekhophi yenombolo yofuzo oluqondiwe.Umthamo wesampula kufanele ucatshangelwe ngesimo sangempela.Yenza ukuhlanjululwa kwesampula ngamanzi angenayo i-nuclease noma i-TE Buffer, uma kunesidingo.
Ukusabela Cizimo
| Inqubo ye-PCR evamile | Inqubo ye-PCR esheshayo | ||||||
| Inqubo | Temp. | Isikhathi | Umjikelezo | Inqubo | Temp. | Isikhathi | Umjikelezo |
| Reverse Transcription | 50℃ | 10-20 imizuzu | 1 | Reverse Transcription | 50℃ | 5 imiz | 1 |
| I-Polymerase Ukwenza kusebenze | 95℃ | 1-5 amaminithi | 1 | I-Polymerase Ukwenza kusebenze | 95℃ | 30s | 1 |
| I-Denaturation | 95℃ | 10-20s | 40-50 | I-Denaturation | 95℃ | 1-3s | 40-50 |
| Anealing futhi Isandiso | 56-64℃ | 20-60s | Anealing futhi Isandiso | 56-64℃ | 3-20s | ||
Ikhwalithi yokulawula
1.Ukutholwa komsebenzi: ukuzwela, ukucaciswa nokuphindaphinda kwe-qPCR.
2.Awukho umsebenzi we-nuclease exogenous: akukho endonuclease exogenous kanye exonuclease ukungcola.
Amanothi
1.Izinga lokukhulisa i-DNA polymerase elisheshayo alikho ngaphansi kuka-1kb/10s.Amathuluzi ahlukene e-PCR anezivinini ezihlukene zokushisisa nokupholisa, izindlela zokulawula izinga lokushisa kanye nokuguquguquka kwe-thermal, futhi ngaleyo ndlela ukuthuthukiswa kokugxilisa kwakho kwe-primer/probe kanye nendlela yokugijima kuhlanganiswe nethuluzi lakho le-PCR elisheshayo kubalulekile.
2.Lo mkhiqizo wenza ukusetshenziswa okubanzi, futhi ulungele ukuxilongwa kwamangqamuzana okuzwela kakhulu.Indlela ye-PCR enezinyathelo ezintathu iyanconywa kuma-primer anezinga lokushisa eliphansi le-anneal noma ukukhulisa izingcezu ezinde ngaphezu kuka-200 bp.
3.Njengoba ama-amplicon ahlukene esebenza ngendlela ehlukile ye-dUTP kanye nokuzwela okuhlukile ku-UNG, ama-reagents kufanele athuthukiswe uma ukuzwela kokutholwa kuncipha lapho kusetshenziswa uhlelo lwe-UNG.Sicela usithinte ukuze uthole usizo lobuchwepheshe uma kudingeka.
4.Ukuze ugweme ukukhuliswa kwemikhiqizo ye-PCR ye-carryover, indawo yokuhlola ezinikele kanye ne-pipette kuyadingeka ukuze kukhuliswe.Sebenzisa ngamagilavu futhi ushintshe njalo futhi ungavuli ishubhu le-PCR ngemva kokukhulisa.
