Ikhithi ye-ForeDirect RT-qPCR
Incazelo yekhithi:
◮Ukuze uthole i-RNA eqondile yokukhulisa isikhathi sangempela kusuka kumaqoqo we-swab ngaphandle kwezinqubo zokuhlanza i-RNA.Le khithi iqeda imijikelezo ye-qRT-PCR phakathi nehora elilodwa.Ingxube ye-enzyme iyinhlanganisela elungiselelwe ye-Reverse Transcriptase, i-Hot-Start Taq DNA polymerase, i-RNase inhibitor.I-Reaction Buffer iqukethe zonke izingxenye ezidingekayo, okuhlanganisa izingxenye zebhafa ezithuthukisiwe, uMg2+, i-dUTP, nama-dNTP.
Incazelo
I-ForeDirect RT-qPCR Kit yakhelwe ukukhulisa isikhathi sangempela kwe-RNA kusuka kumaqoqo we-swab ngaphandle kwezinqubo zokuhlanza i-RNA.Le khithi iqeda imijikelezo ye-qRT-PCR phakathi nehora elilodwa.Ingxube ye-enzyme iyinhlanganisela elungiselelwe ye-Reverse Transcriptase, i-Hot-Start Taq DNA polymerase, i-RNase inhibitor.I-Reaction Buffer iqukethe zonke izingxenye ezidingekayo, okuhlanganisa izingxenye zebhafa ezithuthukisiwe, uMg2+, i-dUTP, nama-dNTP.
Imininingwane
100T
Izingxenye zekhithi
| I-ejenti yokukhululwa kwe-Nucleic acid |
| Isivikeli se-RNA |
| 2× RT-qPCR Buffer |
| Ingxubevange ye-Enzyme(Taq&M-MLV) |
| Iziyalezo |
Uhlelo lokusebenza lwe-PCR
1. Uma amashubhu okusabela e-PCR esenziwe i-centrifuged kafushane, wabeke ethangini lesampula lethuluzi lokukhulisa izwi.
2. Iphrothokholi eshisayo
| Isinyathelo | Izinga lokushisa | Isikhathi | Imijikelezo | |
| 1 | Hlehlisa ukuloba | 50℃ | 15 imiz | 1 |
| 2 | I-pre-denaturation | 95℃ | 1 iminithi | 1 |
| 3 | I-Denaturation | 95℃ | 10 imizuzwana | 42 |
| 4 | I-Anneal/Isandiso | 60℃ | 30 imizuzwana | |
Qaphela:Isignali ye-fluorescent itholwe ngokushesha ngemva kwesinyathelo sokunwetshwa somjikelezo ngamunye.
3. Ngemva kokusetha, gcina ifayela bese usebenzisa uhlelo lokusabela.
Izinyathelo Zokusebenza
Ngaphambi kokulungiswa kwe-reagent, wonke ama-reagents kukhithi kufanele ancibilikiswe ekamelweni lokushisa futhi axutshwe ngobumnene.
Ukulungiswa kwe-reagent
1. Lungiselela i-Nucleic Acid Release Mix
| Isakhi | Ivolumu ngeSampula ngayinye noma Isilawuli |
| I-ejenti yokukhululwa kwe-Nucleic acid | 5μL |
| Isivikeli se-RNA | 0.5 μL |
2. Lungiselela Imiksi Yokusabela
| Isakhi | Ivolumu ngeSampula ngayinye noma Isilawuli |
| 2× RT-qPCR Buffer | 15 μl |
| Ingxubevange ye-Enzyme(Taq&M-MLV) | 1.5 μL |
| Ama-Primers nama-Probes | 1μL |
Isitoreji kanye Nempilo yeshelufu
-Ikhithi kufanele igcinwe ku -20± 5℃.Isikhathi sokuqinisekisa yizinyanga eziyi-12.
-Gwema imijikelezo yokuqhwaza ephindaphindiwe (<5 imijikelezo).
