I-Cell Direct RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready One-step qRT-PCR Kits
Izincazelo
Le khithi isebenzisa isistimu ye-lysis buffer eyingqayizivele engakhulula ngokushesha i-RNA kumasampula eseli akhulisiwe ukuze kusabela i-RT-qPCR, ngaleyo ndlela iqede inqubo yokuhlanza i-RNA edla isikhathi nekhandlayo.Isifanekiso se-RNA singatholakala ngemizuzu engu-7 nje.I-5×I-Direct RT Mix kanye no-2×Ama-reagents aqondile we-qPCR Mix-SYBR ahlinzekwa yikhithi angathola ngokushesha nangempumelelo imiphumela ye-PCR yobuningi besikhathi sangempela.
5×I-Direct RT Mix kanye no-2×I-Direct qPCR Mix-SYBR inokubekezelela okuqinile kwe-inhibitor, futhi i-lysate yamasampuli ingasetshenziswa njengesifanekiso se-RT-qPCR ngokuqondile.Le khithi iqukethe i-RNA high-affinity Foregene reverse transcriptase, kanye ne-Hot D-Taq DNA polymerase, dNTPs, MgCl2, isigcinalwazi sokusabela, i-PCR optimizer nesiqinisi.
Imininingwane
200×20μl Rxns, 1000×20μl rxn
Izingxenye zekhithi
| Ingxenye I | Isivimbeli CL |
| I-Foregene Protease Plus II | |
| Isilondolozi se-ST | |
| Ingxenye II | I-DNA Eraser |
| 5× Imiksi ye-RT eqondile | |
| 2× Direct qPCR Mix-SYBR | |
| I-50 × I-ROX Reference Dye | |
| RNase-Free ddH2O | |
| Iziyalezo | |
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.
Umdwebo
Isitoreji kanye Nempilo yeshelufu
Ingxenye I yale kit 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-QPCR Mix-SYBR eqondile 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.
Isithasiselo 1:Iphakethe lengxenye ye-Cell Direct RT-qPCR Kit
1.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 |
2.RT Imiksi
|
| |
| 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 |
|
| ||
| Izingxenye zekhithi (Isistimu yokusabela engu-20 μl) | I-DRT-01011-C1 | I-DRT-01011-C2 |
| 200 T | 1000 T | |
| 2× Direct qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
| I-50 × I-ROX Reference Dye | 40 ml | 200 μl |
| I-ddH yamahhala ye-RNase2O | 1.7 ml | 10 ml |
Amamanuwali wemiyalo:
