I-Multiplex qPCR Probe Premix
Inombolo yekati: HCB5051A
I-TaqMan multiplex qPCR Master Mix (i-Dye Based) iyisixazululo sangaphambili sokukhuliswa komthamo we-PCR wesikhathi sangempela esingu-2 × esibonakala ngokuzwela okuphezulu nokucaciswa, okuluhlaza okwesibhakabhaka ngombala, futhi kunomphumela wokwengezwa kwesampula.Lo mkhiqizo uyi-2x Mix pre-mixed reagent enika amandla ukusabela kwe-PCR okune-fluorescent quantitative ekuphenduleni okukodwa kahle.Lo mkhiqizo uqukethe indlela ye-antibody eguqulelwe ngofuzo yokuqala i-enzyme ye-Taq, ithuthukisa kakhulu ukuzwela kokukhulisa nokucaciswa kwayo.Ngasikhathi sinye, lo mkhiqizo uthuthukise ngokujulile isibari sokusabela okuningi, esingathuthukisa ukusebenza kahle kokukhulisa ukusabela futhi sikhuthaze ukukhuliswa okusebenzayo kwezifanekiso zokugxila okuphansi.Lo mkhiqizo ungasetshenziselwa ukuhlaziya i-genotyping kanye ne-multiplex.
Ukucaciswa
| Isiqalo Esishisayo | Isiqalo esishisayo esakhelwe ngaphakathi |
| Indlela yokuthola | Ukutholwa kwe-Primer-probe |
| Indlela ye-PCR | qPCR |
| I-Polymerase | Taq DNA polymerase |
| Uhlobo lwesampula | I-DNA |
Izimo Zokugcina
Umkhiqizo uthunyelwa ngeqhwa elomile futhi ungagcinwa ku -25 ~ -15 ℃ iminyaka emi-2.
Iziyalezo
1. UkusabelaUhlelo
| Izingxenye | Ivolumu (μL) | Ukugxila Kokugcina |
| 2× TaqMan multiplex qPCR Master Mix | 12.5 | 1× |
| Ingxube ye-Primer (10 μmol/L) a | × | 0.1 - 0.5 μmol/L |
| Ingxube ye-Probe (10 μmol/L)b | × | 50 - 250 nmol/L |
| Udayi wereferensi we-Rox | 0.5 | 1× |
| I-DNA/cDNA yesifanekiso | 1-10 | - |
| ddH2O | kufika ku-25 | - |
Amanothi:Hlanganisa kahle ngaphambi kokusetshenziswa ukuze ugweme amabhamuza amaningi ekunyakazeni okunamandla.
a.I-Primer concentration: I-Primer Mix iqukethe amapheya amaningi weziqalo, ngokuvamile i-primer ngayinye ekuhlanganiseni kokugcina kuka-0.2 μmol/L futhi ingalungiswa phakathi kuka-0.1 no-0.5 μmol/L ngokufanelekile.
b.Ukugxiliswa kwe-Probe: I-Probe Mix iqukethe ama-probe amaningi anezimpawu ezihlukene ze-fluorescence, futhi ukugxila kwe-probe ngayinye kungalungiswa phakathi kuka-50 no-250 nmol/L ngokuya ngesimo esithile.
1.Ireferensi yodayi we-Rox:Isetshenziswa ethuluzini lokukhulisa i-Real Time PCR njenge-Applied Biosystems ukuze kulungiswe iphutha lesiginali ye-fluorescence ekhiqizwe phakathi kwemithombo;lo mkhiqizo awunayo ireferensi yodayi we-Rox.I-Cas#10200 iyanconywa uma kudingeka.
2.Ukuhlanjululwa kwesifanekiso: I-qPCR izwela kakhulu, futhi kuyanconywa ukuthi kuhlanjululwe isifanekiso ukuze sisetshenziswe.Uma isifanekiso siyisixazululo sesitoko se-cDNA, ivolumu yesifanekiso akumele yeqe u-1/10 wevolumu iyonke.
3.Isistimu yokusabela: 25μL, 30μL noma 50 μL iyanconywa ukuze kuqinisekiswe ukusebenza kahle nokuphindaphinda kokukhulisa isakhi sofuzo okuqondiwe.
4.Ukulungiswa kwesistimu: Sicela ulungiselele ebhentshini elihlanzekile kakhulu, futhi usebenzise amathiphu namashubhu okusabela ngaphandle kwezinsalela ze-nuclease;kunconywa ukusebenzisa amathiphu nge-cartridges yokuhlunga.Gwema ukungcola okuphambanayo kanye nokungcoliswa kwe-aerosol.
2.Uhlelo lokusabela
| Isinyathelo somjikelezo | Temp. | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 95 ℃ | 5 imiz | 1 |
| I-Denaturation | 95 ℃ | 15 isekhondi | 45 |
| I-Annealing/Extension | 60 ℃ | 30 isekhondi |
Amanothi:
1.I-Annealing/Extension: Izinga lokushisa nesikhathi kungalungiswa ngokufanelekile ngokwenani eliklanyelwe le-primer Tm.
2.Ukutholwa kwesiginali ye-Fluorescence: Isikhathi sokutholwa kwesiginali ye-fluorescence esidingekayo kumathuluzi ahlukile wokuthola i-qPCR sihlukile, sicela usethe ngokuya ngomkhawulo wesikhathi omncane.Isikhathi sezinsimbi eziningana ezivamile sisethwe kanje:
20 isekhondi: Applied Biosystems 7700, 7900HT, 7500 Fast
31 isekhondi: Applied Biosystems 7300
32 isekhondi: Applied Biosystems 7500
Amanothi
Sicela ugqoke i-PPE edingekayo, ijazi lelebhu kanye namagilavu, ukuze uqinisekise impilo nokuphepha kwakho!
