Isinyathelo esisodwa Esisheshayo i-RT-qPCR Probe Premix-UNG
Inombolo yekati: HCR5143A
I-One Step RT-qPCR Probe Kit (FOR FAST) iyikhithi yokuthola ngokushesha esekelwe ku-RT-qPCR elungele i-PCR ye-single-plex noma ye-multiplex esebenzisa i-RNA njengesifanekiso (njengegciwane le-RNA).Lo mkhiqizo usebenzisa isizukulwane esisha se-Taq DNA Polymerase eshintshwe amasosha omzimba kanye nesinyathelo esisodwa esinikezelwe i-Reverse Transcriptase, enebhafa ethuthukisiwe yokukhulisa ngokushesha, enesivinini sokukhulisa ngokushesha, ukusebenza kahle kokukhulisa ukuphakama nokucaciswa kwayo.Isekela ukukhulisa okunokulinganisela kukho kokubili i-single-plex kanye ne-multiplex yamasampuli okugxila aphansi naphezulu ngesikhathi esifushane.
Izingxenye
1. 5×RT-qPCR ibhafa (U+)
2. Ingxube ye-enzyme (U+)
Amanothi:
a.I-5×RT-qPCR buffer (U+) ihlanganisa i-dNTP ne-Mg2+;
b.Ingxube ye-enzyme (U+) ihlanganisa i-reverse transcriptase, i-Hot Start Taq DNA polymerase, i-RNase inhibitor ne-UDG;
c.Sebenzisa amathiphu e-RNase-Free, amashubhu e-EP, njll.
Ngaphambi kokusebenzisa, hlanganisa kahle i-5×RT-qPCR buffer (U+).Uma kukhona imvula ngemva kokuncibilika, linda isigcinalwazi sibuyele ekamelweni lokushisa, xuba futhi uhlakaze, bese ulisebenzisa ngokujwayelekile.
Izimo Zokugcina
Umkhiqizo uthunyelwa ngeqhwa elomile futhi ungagcinwa ku -25 ~ -15 ℃ unyaka ongu-1.
Iziyalezo
1. Ukusabela Uhlelo
| Izingxenye | Umthamo (20 μL ukusabela) |
| 2 ×RT-qPCR ibhafa | 4μL |
| Ingxube ye-enzyme (U+) | 0.8μL |
| I-Primer Forward | 0.1~ 1.0μM |
| I-Primer Reverse | 0.1~ 1.0μM |
| I-TaqMan Probe | 0.05~0.25μM |
| Isifanekiso | X μL |
| Amanzi Angenayo I-RNase | kufika ku-25μL |
Amanothi: Ivolumu yokusabela ngu-10-50μL.
2. I-Cycling Protocol (Sejwayelekile)
| Umjikelezo isinyathelo | Temp. | Isikhathi | Imijikelezo |
| Reverse Transcription | 55 ℃ | 10 imiz | 1 |
| I-Denaturation Yokuqala | 95 ℃ | 30 isekhondi | 1 |
| I-Denaturation | 95 ℃ | 10 isekhondi | 45 |
| I-Annealing/Extension | 60 ℃ | 30 isekhondi |
| I-Cycling Protocol (Shesha) Umjikelezo isinyathelo |
Temp. |
Isikhathi |
Imijikelezo |
| Reverse Transcription | 55 ℃ | 5 imiz | 1 |
| I-Denaturation Yokuqala | 95 ℃ | 5 s | 1 |
| I-Denaturation | 95 ℃ | 3 s | 43 |
| I-Annealing/Extension | 60 ℃ | 10 s |
Amanothi:
a.Izinga lokushisa lokuhlehla lokuloba liphakathi kuka-50℃ kuya ku-60℃, ukukhulisa izinga lokushisa kusiza ukukhulisa izakhiwo eziyinkimbinkimbi nezifanekiso zokuqukethwe ze-CG eziphezulu;
b.Izinga lokushisa elilungile le-anneal lidinga ukulungiswa ngokusekelwe kunani le-Tm le-primer, bese ukhetha isikhathi esifushane kakhulu sokuqoqwa kwesignali ye-fluorescence ngokusekelwe ethuluzini le-PCR Lesikhathi Sangempela.
Amanothi
Sicela ugqoke i-PPE edingekayo, ijazi lelebhu kanye namagilavu, ukuze uqinisekise impilo nokuphepha kwakho!
