I-2×Sensi Direct Premix-UNG (Probe qPCR)
Inombolo yekati: HCB5151A
I-SensiDirect Premix-UNG (Probe qPCR) yakhelwe ukwenza i-PCR ngokuqondile ngamasampula ngaphandle kokukhipha i-DNA noma ukulungiswa kwesampula.Lesi sithako siqukethe i-DNA polymerase eqala ukushisa, i-uracil DNA glycosylase (UNG), i-RNase Inhibitor, i-MgCl.2, ama-dNTP (nge-dUTP esikhundleni se-dTTP), neziqinisi, ze-quantitative PCR (qPCR).Lesi sikhungo sithuthukisa ukubekezelela okuphezulu kwe-inhibitor, futhi ngaleyo ndlela singasetshenziswa ngokuqondile ekutholweni kwamasampula afana ne-swab yomphimbo, amathe, igazi eliphelele elilwa nokujiya, i-plasma, ne-serum ngaphandle kokukhipha i-DNA.I-reagent isebenzisa isivikelo sobunikazi se-qPCR nama-enzyme axubile e-anti-inhibitory DNA polymerase kanye ne-UNG enzyme.Ngakho-ke, ingathola ukukhuliswa okuhle kwezakhi zofuzo eziqondiwe kumasampuli aqukethe ama-inhibitor futhi ivimbele ukukhulisa okungamanga okubangelwa ukungcoliswa kwe-PCR kanye ne-aerosol.Lesi sici sihambisana nezinsimbi eziningi ze-PCR ze-fluorescent, njenge-Applied Biosystems, i-Eppendorf, i-Bio-Rad, i-Roche njalonjalo.
Izingxenye
1. 50×I-SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect 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.
I-Cycling Protocol
| Isinyathelo | Izinga lokushisa | Isikhathi | Umjikelezo |
| Ukugaya ukudla | 50℃ | 2 imiz | 1 |
| Ukusebenza kwe-Polymerase | 95℃ | 1-5 iminithi | 1 |
| I-Denature | 95℃ | 10-20s | 40-50 |
| I-Annealing/Extension | 56-64℃ | 20-60s |
Imiyalo yokushaya amapayipi
| I-Reagent | Ivolumu ngayinye ukusabela | Ivolumu ngokusabela ngakunye | Ukugxila Kokugcina |
| I-2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| 25×Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Isampula3, 4 | - | - | - |
| ddH2O | - | - | - |
| Ivolumu ephelele | 25 μl | 50 μL | - |
1. Ukugxila kokugcina kwe-primer kuvame ukuba ngu-0.2μM.Ukuze uthole imiphumela engcono, ukugxiliswa kwe-primer kungenziwa kahle phakathi kwebanga lika-0.2-1μM.
2. Ngokuvamile, ukugxila kwe-probe kungenziwa kahle phakathi kwebanga lika-0.1-0.3μM.Ukugxila okuphelele kophenyo kuhlobene nethuluzi lesikhathi sangempela lokukhulisa i-PCR, uhlobo lophenyo, kanye nohlobo lwento yokulebula ye-fluorescent.Sicela ubheke imanuwali yensimbi noma izimfuneko ezithile zokuhlola ngakunye kwe-fluorescent.
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 ngokungeza amanzi angenayo i-nuclease noma i-TE Buffer, uma kunesidingo.
4. Ivolumu enconyiwe yamasampuli ahlukene:
| Isampula | Ivolumu eyodwa 50 μL ukusabela | Ubuningi isilinganiso |
| Igazi eligcwele i-Anticoagulated | 2.5 μL | 5% |
| I-Plasma | 15 μl | 30% |
| I-Serum | 10 μL | 20% |
| I-swab yomphimbo | 10 μL | 20% |
| Amathe | 10 μL | 20% |
Ikhwalithi yokulawula
1. Ukutholwa komsebenzi: ukuzwela, ukucaciswa nokuphindaphinda kwe-qPCR.
2. Awukho umsebenzi we-nuclease exogenous: akukho endonuclease exogenous kanye exonuclease ukungcola.
Amanothi Omkhiqizo
1. Lo mkhiqizo usebenzisa uhlobo lwenoveli lwe-polymerase ye-DNA eqala ukushisa, engenziwa isebenze emizuzwini engu-1-5.Njengoba isilondolozi sawo sokusabela sesithuthukisiwe, sifaneleka kakhulu ku-PCR yobuningi be-fluorescence ephindwe kabili noma eminingi kusetshenziswa indlela yokuhlola.
2. Uma inani le-Rn lokukhulisa i-PCR liphansi kakhulu noma ukukhulisa ngokusobala kuvinjiwe, ukwehlisa inani lesampula, ukukhulisa ivolumu yokusabela noma ukuhlanjululwa kwangaphambili kwesampula kungase kuthuthukise imiphumela.
3. Ukuqoqwa kwegazi, amathe, umchamo, i-swab yomphimbo, njll. kufanele kulandele izimfuneko zenqubo yomtholampilo, futhi isampula elisha lingasetshenziswa ukuvimbela ukuwohloka kwe-nucleic acid.
4. Njengoba ama-amplikhoni ahlukene enokusebenza kahle okuhlukile kokusetshenziswa ku-dUTP nokuzwela ku-UNG, ama-reagents kufanele athuthukiswe uma ukuzwela kokutholwa kuncipha uma kusetshenziswa uhlelo lwe-UNG.Sicela usithinte ukuze uthole usizo lobuchwepheshe uma kudingeka.
5. Ukuze ugweme ukukhuliswa kwemikhiqizo ye-PCR ye-carryover phakathi kokusabela kwesinyathelo esisodwa, indawo yokuhlola ezinikele kanye ne-piette kuyadingeka ukuze kukhuliswe.Sebenzisa ngamagilavu futhi ushintshe njalo futhi ungavuli ishubhu lokusabela ngemva kokukhulisa i-PCR.
