(96-lelei) QuickEasy Cell Direct RT-qPCR Kit – SYBR Green I
Fa'amatalaga
O le(96-lelei) QuickEasyTM Cell Direct RT-qPCR Kit–SYBR Green Isauniase faiga fa'apipi'i lysis tulaga eseto vave tatala RNAmai sela fa'aa'efaʻataʻitaʻiga mo faʻataʻitaʻiga RT-qPCR, faʻaumatia le taimi faʻaalu ma le galueRNA fa'amamāina fa'agasologa, ma e na'o le 7 minute e maua ai le fa'ata'ita'iga RNA mana'omia.O le5× Direct RT Mixma2× Direct qPCR Mix-SYBRsaunia i totonu o le pusa e mafai ona vave mamataalia maua taimi moni quantitativeI'uga PCR.
5 × Direct RT Mix ma 2 × Direct qPCR Mix-SYBR o loʻo i ai le faʻapalepale malosi o le inhibitor, ma e mafai ona faʻaogaina le lysate o le faʻataʻitaʻiga e faʻataʻitaʻiina e fai ma faʻataʻitaʻiga mo le lelei o le faʻaliliuga faʻaliliu ma le faʻalauteleina faʻapitoa.O le reagent o loʻo i ai le Foregene tulaga ese Reverse Transcriptase faʻatasi ai ma le maualuga maualuga mo le RNA, faʻapea foʻi ma le Hot D-Taq DNA Polymerase, dNTPs, MgCl2 , tali faʻafefe, PCR optimizer ma stabilizer, ma e mafai ona faʻaogaina faʻatasi ma le lysis buffer e iloa ai le faʻataʻitaʻiga vave, faigofie ma saʻo, ma o loʻo i ai uiga o le maualuga maualuga, malosi faʻapitoa ma le mautu lelei.
O le pusa o loʻo faʻatatau i le micro-system lysis o 96-well cultured cell, ma e lelei le tutusa ma le tumau;o vaega kit e maua ai 96 lysis reactions, 96 reverse transcription reactions ma 96 × 2 qPCR reactions, lea e mafai ona fetaui ma le 96-well plate plate mo le faʻaaogaina e tasi, aloese mai le faʻaleagaina e mafua mai i le tatalaina faifaipea, faʻamalo ma thawing o reagents ma le faʻaleagaina o le faʻatinoga o gaioiga.
Vaega o pusa
| Fausia pusa ( 50 μL lysis system/20 μLRT faiga tali atu /20μL qPCR faiga tali) | DRT-03011 | Fa'amatalaga | |
| 96T | |||
| VaegaI | PuipuiCL | 5 ml | selaLysis |
| ForegeneProtease Plus II | 100 μL | ||
| PuipuiST | 500 μL | ||
| Vaega II | DNATiipe | 100 μL | |
| 5× Direct RT Mix | 400 μL | RT | |
| 2× Faafefiloi qPCR tuusao-SYBR | 1 mL × 2 | qPCR | |
| 50× ROX Fa'ailoga Fa'ailoga | 400µL | ||
| RNase- Sa'olotoddH2 O | 1.7ml |
| |
| Mfaaletausaga | 1 fasi | 1 tautua | |
*: O le lysis reagent DNA Eraser o loʻo aofia i le Vaega II o le pusa;O vaega o le Cell Lysis, RT, ma le qPCR e mafai ona fa'atau eseese.
Fa'apitoa & itu lelei
■Faigofie ma aoga : faʻatasi ai ma le Cell Direct RT tekinolosi, RNA faʻataʻitaʻiga e mafai ona maua i totonu o le 7 minute.
■ O le faʻataʻitaʻiga manaʻoga e laʻititi, e pei o le 10 sela e mafai ona suʻeina.
■ Tulaga maualuga: e mafai ona vave iloa le RNA i sela o loʻo faʻatupuina ile 384, 96, 24, 12, 6-well plates.
■ DNA Eraser e mafai ona vave aveese genomes tatala, faʻaitiitia le aʻafiaga i faʻataʻitaʻiga mulimuli ane.
■ Optimized RT ma qPCR system e sili atu ona lelei le fa'aliliuina o le RT-PCR la'asaga e lua ma sili atu ona patino le PCR, ma sili atu ona fa'asa'o ile fa'ainitaneti tali ole RT-qPCR.
Fa'aoga pusa
Avanoa o le faʻaogaina: sela faʻaleaganuʻu.
- RNA fa'amatuu mai ile fa'ata'ita'iga lysis: na'o le fa'ata'ita'iga RT-qPCR o lenei pusa.
- O le pusa e mafai ona faʻaoga mo faʻamoemoega nei: suʻesuʻega faʻamatalaga o kenera, faʻamaoniga o le siRNA-mediated gene silencing effect, suʻesuʻeina o fualaau, ma isi.
Teuina ma le ola fa'asao
Vaega I o lenei pusa e tatau ona teuina i le 4℃;Vaega II e tatau ona teuina ile -20℃.
Foregene Protease Plus II e tatau ona teuina ile 4℃, aua le aisa ile -20℃.
Reagent 2×Direct qPCR Mix-Taqman e tatau ona teuina ile -20℃i le pogisa;afai e faʻaaoga soo, e mafai foi ona teuina i le 4℃ mo le teuina mo taimi pupuu (fa'aoga i totonu ole 10 aso).
Taimi Moni PCR fa'avae mamanu fa'avae
Fa'aimua Fa'asaga i luma ma Fa'ailoga Fa'asaga
Mo PCR Taimi Moni, e taua tele le mamanu muamua.Primers e fesoʻotaʻi ma le faʻapitoa ma le lelei o le PCR faʻalauteleina, ma e mafai ona mamanuina e faʻatatau i mataupu faavae nei:
- Ulua'i umi: 18-30bp.
- GC anotusi: 40-60%.
- Tm tau: Polokalama fa'ailoga muamua, pei ole Primer 5, e mafai ona tu'uina atu le tau Tm ole fa'avae.Ole tau Tm ole pito i luga ma lalo e tatau ona latalata ile mea e mafai ai.E mafai foi ona fa'aoga le fua fa'atatau Tm: Tm = 4 °C (G + C) + 2 °C (A + T).Pe a faia le PCR, o le vevela i lalo ifo o le tau Tm muamua o le 5 °C e masani ona filifilia e avea ma vevela faʻapipiʻi (o le siʻitia tutusa i le vevela faʻafefe e mafai ona faʻateleina le faʻapitoa o le tali PCR).
- Primers ma PCR oloa:
- Fuafuaga muamua PCR amplification umi o oloa e sili ona lelei 100-150bp.
- E tatau ona 'alo'ese mai mea fa'apitoa i le vaega lona lua o le fa'ata'ita'iga i le tele e mafai ai.
- Aloese mai le fa'avaeina o fa'avae fa'aopoopo e 2 pe sili atu i le va o pito 3' ole pito i luga ma lalo.
- Primer 3′ e le mafai ona iai ma le 3 fa'aopoopo G po'o le C.
- E le mafai ona iai ni fausaga fa'aopoopo i le primers, a leai o le a fausia se fausaga lauulu, e a'afia ai le fa'alauteleina o le PCR.
- ATCG e tatau ona tufatufa tutusa tutusa i le faasologa muamua, ma le 3′ fa'avae pito e tatau ona aloese mai le T.
Fa'aopoopo1:Cell DirectRT-qPCR Pusa vaegat pusa faaopoopo
1.Cell Lysis Fofo
| Cell Lysis Fofo | |||
| Vaega o pusa (24-vaieli lysis system / vaieli) | DRT-01011-A1 | DRT-01011-A2 | |
| 100 T | 500 T | ||
| VaegaI | Puipui CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Puipui ST | 1 ml × 2 | 10 ml | |
| VaegaII | DNA tape | 400 μl | 1 ml × 2 |
| RT Mix | |
| Vaega o pusa (20 μl faiga tali) | DRT-01011-B1 |
| 200 T | |
| 5× Direct RT Mix | 800 μl |
| RNase-Free ddH2O | 1.7 ml × 2 |
| qPCR Mix | ||
| Vaega o pusa (20 μl faiga tali) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2× Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
| 20× ROX Fa'ailoga Fa'ailoga | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Tusi Taiala:
