Pusa RT qPCR Direct Direct—SYBR GREEN I Direct Cell Lysis Cell Ready Pusa qRT-PCR e Tasi Laasaga
Fa'amatalaga
O lenei pusa o loʻo faʻaaogaina se faiga faʻapipiʻi lysis faʻapitoa e mafai ona vave faʻasaʻo le RNA mai faʻataʻitaʻiga faʻaleaganuʻu mo le RT-qPCR tali, ma faʻaumatia ai le taimi faʻaalu ma le galue RNA faʻamamaina.E mafai ona maua le mamanu RNA ile na'o le 7 minute.O le 5×Direct RT Mix ma le 2×Tu'usa'o le qPCR Mix-SYBR reagents e saunia e le pusa e mafai ona vave ma lelei le mauaina o fa'ai'uga PCR fa'atusatusa i le taimi moni.
5×Direct RT Mix ma le 2×Direct qPCR Mix-SYBR o loʻo i ai le faʻapalepale malosi o le inhibitor, ma o le lysate o faʻataʻitaʻiga e mafai ona faʻaaogaina e fai ma faʻataʻitaʻiga mo RT-qPCR saʻo.O lenei pusa o loʻo i ai le RNA maualuga-affinity Foregene reverse transcriptase, ma Hot D-Taq DNA polymerase, dNTPs, MgCl2, tali fa'afefete, PCR optimizer ma fa'amautu.
Fa'amatalaga
200×20μl Rxns, 1000×20μl Rxns
Vaega o pusa
| Vaega I | Puipui CL |
| Foregene Protease Plus II | |
| Puipui ST | |
| Vaega II | DNA tape |
| 5× Direct RT Mix | |
| 2× Direct qPCR Mix-SYBR | |
| 50× ROX Fa'ailoga Fa'ailoga | |
| RNase-Free ddH2O | |
| Faatonuga | |
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.
Ata
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-SYBR 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'aopoopoga 1: Fa'aputuga fa'aopoopo vaega o le Kit RT-qPCR
1.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 |
2.RT Faafefiloi
|
| |
| 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 |
|
| ||
| Vaega o pusa (20 μl faiga tali) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T | 1000 T | |
| 2× Direct qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
| 50× ROX Fa'ailoga Fa'ailoga | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Tusi Taiala:
