(96-well) QuickEasy Cell Direct RT-qPCR Kit – Taqman
Izincazelo
I(96-kahle) QuickEasyTM I-Cell Direct RT-qPCR Kit-Taqmaninikezaisistimu ye-lysis buffer eyingqayiziveleto khulula ngokushesha i-RNAkusuka kuseli elikhulisiweamasampula wokusabela kwe-RT-qPCR, eqeda isikhathi esiningi futhi ekhandlayoInqubo yokuhlanza i-RNA, futhi kuthatha imizuzu engu-7 kuphela ukuthola ithempulethi ye-RNA edingekayo.I5× Imiksi ye-RT eqondilefuthi2× Direct qPCR Mix-SYBRenikeziwe ebhokisini can ngokushesha futhithola ngempumelelo ubuningi besikhathi sangempelaImiphumela ye-PCR.
I-5× Direct RT Mix kanye ne-2× Direct qPCR Mix-Taqman inokubekezelela okuqinile kwe-inhibitor, futhi ingasebenzisa i-lysate yesampula ukuze ihlolwe njengesifanekiso sokuloba okubuyela emuva okuphumelelayo kanye nokukhulisa okuthile.I-reagent iqukethe i-Foregene unique Reverse Transcriptase ehlobene kakhulu ne-RNA, kanye ne-Hot D- Taq DNA Polymerase, i-dNTPs, i-MgCl2 , i-react buffer, i-PCR optimizer ne-stabilizer, futhi ingasetshenziswa ngokuhambisana ne-lysis buffer ukuze ibone isampula ngokushesha, kalula futhi ngokunembile, futhi inezici zokuzwela okuphezulu, ukucaciswa okuqinile nokuzinza okuhle.
Ikhithi ihloselwe i-micro-system lysis yamaseli akhuliswe kahle angama-96, futhi inokufana okuhle nokuvumelana;izingxenye zekhithi zinikeza ukusabela kwe-96 lysis, 96 reverse transcription reactions kanye 96×2 qPCR reactions, ezingahlangabezana 96-well cell plate ukusetshenziswa ngesikhathi esisodwa, ukugwema ukungcola okubangelwa ukuvulwa okuphindaphindiwe, ukubanda nokuncibilika kwama-reagents kanye nokonakala kokusebenza kwe-reagent.
Izingxenye zekhithi
| Ukwakheka kwekhithi ( 50 μUhlelo lwe-L lysis/20 mlRT uhlelo lokusabela /20μL qPCR uhlelo lokusabela) | I-DRT-03021 | Phawula | |
| 96T | |||
| IngxenyeI | IbhafaCL | 5 ml | IseliLysis |
| I-ForegeneI-Protease Plus II | 100 μL | ||
| IbhafaST | 500 μL | ||
| Ingxenye II | I-DNAIsisuli | 100 μL | |
| 5× Imiksi ye-RT eqondile | 400 μL | RT | |
| 2× Imiksi eqondile ye-qPCR-Taqaman | 1 mL × 2 | qPCR | |
| I-50 × I-ROX Reference Dye | 400µL | ||
| RNase- MahhaladdH2 O | 1.7mL |
| |
| Munyaka wonke | 1 ucezu | 1 ukukhonza | |
*: I-lysis reagent DNA Eraser ifakiwe Engxenyeni II yekhithi;I-Cell Lysis, RT, kanye nezingxenye ze-qPCR zingathengwa 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:
