I-Cell Direct RT qPCR Kit—Taqman Direct Cell Lysis Cell Ready Isinyathelo esisodwa se-qRT-PCR Kits Probe
Izincazelo
Lo mkhiqizo usebenzisa isistimu ye-lysis buffer eyingqayizivele ukuze ukhulule ngokushesha i-RNA kumasampula eseli akhulisiwe ukuze kusabela i-RT-qPCR, uqede inqubo yokuhlanza i-RNA ethatha isikhathi futhi ekhandlayo, kanye nemizuzu engu-7 kuphela ukuthola ithempulethi edingekayo ye-RNA, nge-5× Direct RT Mix, 2× Direct qPCR Mix-Taqman thola imiphumela ye-qualyntitive PCR ngokushesha nangendlela efanele.
I-5× Direct RT Mix kanye ne-2× Direct qPCR Mix-Taqman zinokubekezelela okuqinile kwe-inhibitor futhi zingenza ukuguqulwa okuphumelelayo nokukhulisa ukukhulisa okuthile kusetshenziswa i-lysate yesampula ukuze ikalwe njengesifanekiso.I-reagent iqukethe i-Foregene Reverse Transcriptase, i-Hot D-Taq DNA Polymerase, i-dNTPs, i-MgCl2, I-Reaction Buffer, i-PCR Optimizer ne-Stabilizer, engasetshenziswa ne-lysis buffer ukuthola ngokushesha futhi kalula amasampula, futhi inezici zokuzwela okuphezulu, ukucaciswa nokuzinza.
Imininingwane
200×20μl Rxns, 1000×20μl rxn
Izingxenye zekhithi
QuickEasyTMI-Cell Direct RT-qPCR Kit-Taqman | ||||
Izingxenye zekhithi 20μl qPCR Reaction System | I-DRT-01021 | I-DRT-01022 | Qaphela | |
200 T | 1000 T | |||
Ingxenye I | Isivimbeli CL | 4 ml | 20 ml | Iseli Lysis |
I-Foregene Protease Plus II | awu 80l | 400 μl | ||
Isilondolozi se-ST | 400 μl | 1 ml × 2 | ||
Ingxenye II | I-DNA Eraser | awu 80l | 400 μl | |
5×Imiksi ye-RT eqondile * | 160 μl | 800 μl | RT | |
2× Direct qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
I-20 × ROX Ireferensi Idayi | 40 ml | 200 μl | ||
I-ddH yamahhala ye-RNase2O | 1.7 ml | 10 ml | ||
Incwadi Yeziqondiso | 1 isiqephu | 1 isiqephu |
*:I-Cell Lysis, 5×Direct RT Mix, 2× Direct qPCR Mix-Taqman angathengwa ngokuhlukana
Izici&izinzuzo
■Kulula futhi kusebenza ngempumelelo : Ngobuchwepheshe be-Cell Direct RT, amasampula e-RNA angatholwa ngemizuzu engu-7 nje.
■ Isidingo sesampula sincane, siphansi njengoba amaseli ayi-10 angahlolwa.
■ Ukuphuma okuphezulu: ikwazi ukubona ngokushesha i-RNA kumaseli akhiwe kumapuleti angu-384, 96, 24, 12, 6-wemithombo.
■ I-DNA Eraser ingasusa ngokushesha ama-genome akhululiwe, inciphise kakhulu umthelela emiphumeleni yokuhlola elandelayo.
■ Isistimu ye-RT ethuthukisiwe kanye ne-qPCR yenza ukuloba okuhlanekezela okuzinyathelo ezimbili kwe-RT-PCR kusebenze kahle futhi i-PCR icace kakhulu, futhi imelane kakhulu ne-RT-qPCR reaction inhibitors.
Isicelo sekhithi
Ububanzi bokusebenza: amaseli akhulisiwe.
- I-RNA ekhishwe ngesampula ye-lysis: isebenza kuphela kusifanekiso se-RT-qPCR sale kit.
- Ikhithi ingasetshenziselwa lezi zinhloso ezilandelayo: ukuhlaziywa kwesisho sofuzo, ukuqinisekiswa komphumela wokuthulisa ufuzo we-siRNA-mediated, ukuhlolwa kwezidakamizwa, njll.
Isitoreji kanye Nempilo yeshelufu
Ingxenye I yale khithi kufanele igcinwe ku-4℃;Ingxenye II kufanele igcinwe ku -20 ℃.
I-Foregene Protease Plus II kufanele igcinwe ku-4℃, ungafrizi ku -20℃.
I-Reagent 2×I-Direct qPCR Mix-Taqman kufanele igcinwe ku -20℃ebumnyameni;uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ ukuze ugcine isikhathi esifushane (sebenzisa phakathi kwezinsuku eziyi-10).
Izimiso ze-Real Time PCR primer design
Dlulisela phambili i-Primer kanye ne-Primer Reverse
Nge-Real Time PCR, ukuklama kokuqala kubaluleke kakhulu.Ama-Primers ahlobene nokucaciswa nokusebenza kahle kokukhulisa i-PCR, futhi angaklanywa ngokubhekiselwa kule migomo elandelayo:
- Ubude be-Primer: 18-30bp.
- Okuqukethwe kwe-GC: 40-60%.
- Inani le-Tm: Isofthiwe yokuklama i-Primer, njenge-Primer 5, inganikeza inani le-Tm le-primer.Amanani e-Tm okuqalisa akhuphuka nomfula kufanele abe seduze ngangokunokwenzeka.Ifomula yokubala ye-Tm nayo ingasetshenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Lapho kwenziwa i-PCR, izinga lokushisa elingaphansi kwenani lokuqala le-Tm elingu-5 °C livame ukukhethwa njengezinga lokushisa lokukhipha isisu (ukwenyuka okuhambisanayo kwezinga lokushisa le-anneal kungakhuphula ukucaciswa kokusabela kwe-PCR).
- Ama-Primers kanye nemikhiqizo ye-PCR:
- I-design primer PCR ubude bomkhiqizo wokukhulisa u-100-150bp.
- Iziqalo zokuklama endaweni yesibili yesakhiwo sesifanekiso kufanele zigwenywe kakhulu ngangokunokwenzeka.
- Gwema ukwakheka kwezisekelo ezi-2 noma ngaphezulu ezihambisanayo phakathi kwamaphethelo angu-3′ weziqalisi ezikhuphuka nomfula.
- Isisekelo setheminali ye-Primer 3′ asikwazi ukuba khona nezinye ezingu-3 ezengeziwe ezilandelanayo ze-G noma C.
- Ama-primer ngokwawo awakwazi ukuba nezakhiwo ezihambisanayo, ngaphandle kwalokho kuzokwakhiwa isakhiwo se-hairpin, esithinta ukukhuliswa kwe-PCR.
- I-ATCG kufanele isatshalaliswe ngokulinganayo ngangokunokwenzeka ekulandeleni kokuqala, futhi isisekelo setheminali esingu-3′ kufanele sigwenywe njengo-T.
Isithasiselo1:Cethi DirectI-RT-qPCR Ingxenye yekhithit iphakethe le-supplement
1.Cell Lysis Solution
Cell Lysis Solution | |||
Izingxenye zekhithi (Isistimu ye-lysis ye-24 / kahle) | I-DRT-01011-A1 | I-DRT-01011-A2 | |
100 T | 500 T | ||
IngxenyeI | Isivimbeli CL | 20 ml | 100 ml |
I-Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
Isilondolozi se-ST | 1 ml × 2 | 10 ml | |
IngxenyeII | I-DNA Eraser | 400 μl | 1 ml × 2 |
I-RT Mix | |
Izingxenye zekhithi (Isistimu yokusabela engu-20 μl) | I-DRT-01011-B1 |
200 T | |
5× Imiksi ye-RT eqondile | 800 μl |
I-ddH yamahhala ye-RNase2O | 1.7 ml × 2 |
I-qPCR Mix | ||
Izingxenye zekhithi (Isistimu yokusabela engu-20 μl) | I-DRT-01021-C1 | I-DRT-01021-C2 |
200 T | 1000 T | |
2× Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
I-20 × I-ROX Reference Dye | 40 ml | 200 μl |
I-ddH yamahhala ye-RNase2O | 1.7 ml | 10 ml |
Amamanuwali wemiyalo: