I-Multiplex One Step RT-qPCR Premix
Incazelo
Inombolo yekati: HCR5141A
I-Multiplex One Step RT-qPCR Premix iyi-multiplex quantitative PCR Kit esekelwe ku-RNA njengesifanekiso.Esivivinyweni, ukuloba okuhlanekezelwe kanye ne-PCR yobuningi kwenziwa ngeshubhu yokusabela efanayo, okwenza umsebenzi wokuhlola ube lula futhi kuncishiswe ubungozi bokungcola.Idizayini eyingqayizivele ye-buffer ne-enzyme mix ingasetshenziswa ohlelweni olulodwa lwe-lyophilized.Ikhithi isebenzisa i-Reverse Transcriptase ekwazi ukumelana nokushisa ukuze kuhlanganiswe kahle i-cDNA yomucu wokuqala kusetshenziswa i-hotstart Taq DNA Polymerase ukuze kukhuliswe ubuningi.Iqukethe isilondolozi sokusabela esilungiselelwe, ukuxubana kwama-enzyme njll., kanye nezici ezivimbela ngempumelelo ukukhulisa i-PCR okungaqondile kanye nokuthuthukisa ukusebenza kahle kokukhulisa ukusabela kwe-qPCR okuningi kwengeziwe, okuvumela ukukhulisa ubuningi be-fluorescence ngenkathi kuqinisekiswa ukusebenza kahle kokukhulisa iziqalo.
Izingxenye
| Igama |
| 1. I-Lyo-Buffer |
| 2. I-Lyo-Enzyme Mix |
| 3. Umvikeli weLyo |
I-condit yezokuthuthaion
A: I-Lyo-buffer nesivikelo: -25~-15℃, impilo yeshalofu unyaka ongu-1.
B: Ingxube ye-Lyo-enzyme, 2-8 ℃, impilo yeshalofu yizinyanga eziyisi-6.
Umyalelo wokusebenza
1. Uhlelo Lokusabela (Thatha u-25μL njengesibonelo)
| Izingxenye | Ivolumu (μL) | Ukugxila Kokugcina |
| I-Lyo-Buffer | 6 | 1* |
| I-Lyo-Enzyme Mix | 1 | - |
| I-Lyo-Protectant | 8 | - |
| I-Primer Mix (10μM) | 1 | 0.1-1uM |
| I-Probe Mix (10μM) | 0.5 | 0.05-0.5uM |
| Ithempulethi ye-RNA | 5 | - |
| I-DEPC H2O | Kufika ku-25 | - |
2. I-Optimized Cycling Protocol
1) Iphrothokholi Ejwayelekile Yokuhamba Ngebhayisikili
| Isiteji sokusabela | Izinga lokushisa | Isikhathi | Umjikelezo | |
| 1 | Hlehlisa ukuloba | 50°Ca | 10 imiz | 1 |
| 2 | I-denaturation yokuqala | 95°C | 5 imiz | 1 |
| 3 | Ukusabela kokukhulisa | 95°C | 15 isekhondi | 45 imijikelezo |
| 60°Cb | 30 isekhondic |
2) Fast Cycling Protocol
|
| Isiteji sokusabela | Izinga lokushisa | Isikhathi | Umjikelezo |
| 1 | Hlehlisa ukuloba | 50°Ca | 2 imiz | 1 |
| 2 | I-denaturation yokuqala | 95°C | 2 isekhondi | 1 |
| 3 | Ukusabela kokukhulisa | 95°C | 1 isekhondi |
45 imijikelezo |
| 60°Cb | 13 isekhondic |
Qaphela:
a) Reverse okulotshiweyo: Izinga lokushisa lingakhetha u-42°C noma u-50°C imizuzu engu-10-15.
b) Ukusabela kokukhulisa: Izinga lokushisa lilungiswa ngokuvumelana nenani le-Tm lama-primer aklanyelwe.
c)I-Fluorescence isignali ukutholwa: Sicela usethe inqubo yokuhlola ngokwezidingo ze-imanuwali yensimbi.
Ulwazi Lobuchwepheshe/Imininingwane
| Isiqalo Esishisayo | Isiqalo esishisayo esakhelwe ngaphakathi |
| Indlela yokuthola | Ukutholwa kwe-Primer-probe |
| Indlela ye-PCR | Isinyathelo esisodwa RT-qPCR |
| Uhlobo lwesampula | I-RNA |
Amanothi
1. Lo mkhiqizo owokusetshenziswa ucwaningo kuphela.
2. Sicela ugqoke i-PPE edingekayo, ijazi lelebhu kanye namagilavu, ukuze uqinisekise impilo yakho nokuphepha!
