O loʻo i ai se uiga lelei ma alualu i luma i le fiafia o tagata faʻatau, o loʻo faʻaleleia pea e le matou faʻalapotopotoga a matou oloa lelei e faʻamalieina ai manaʻoga o tagata faʻatau ma sili atu ona taulaʻi i le saogalemu, faʻamaoni, faʻamatalaga o le siosiomaga, ma le faʻafouina o Factory Supply China Convenient and Quick Rt-PCR Dspathtm 4X One-Step Multiplex Master Mix, O la matou aufaigaluega lavelave e mafai ona lagolagoina ma le loto atoa.Matou te faʻafeiloaʻi ma le faʻamaoni ia te oe e mautinoa e te tu i la matou upega tafaʻilagi ma kamupani ma meli mai ia i matou lau suʻesuʻega.
O loʻo i ai se uiga lelei ma alualu i luma i le fiafia o tagata faʻatau, o loʻo faʻaleleia pea e le matou faʻalapotopotoga a matou oloa lelei e faʻamalieina manaʻoga o tagata faʻatau ma faʻalautele atili i le saogalemu, faʻamaoni, faʻamatalaga o le siosiomaga, ma le faʻafouina oSaina Taq DNA Polymerase, Qpcr, O loʻo i ai nei a matou fofo sili ona lelei ma faʻatauga faʻapitoa ma vaega faʻapitoa. Faatasi ai ma le atinaʻeina o la matou kamupani, e mafai ona matou tuʻuina atu i tagata faʻatau oloa sili ona lelei, lagolago faʻapitoa lelei, auaunaga atoatoa pe a uma ona faʻatau.

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×Direct qPCR Mix-SYBR reagents e saunia e le pusa e mafai ona vave ma lelei ona maua i'uga o le PCR fa'atusatusa i le taimi moni.

5 × Direct RT Mix ma 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

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 i le 4℃, aua le fa'aisa ile -20℃.

Reagent 2×Direct qPCR Mix-SYBR e tatau ona teuina i le -20 ℃ i le pogisa;pe a faʻaaoga soo, e mafai foi ona teuina i le 4 ℃ mo le teuina mo taimi pupuu (faʻaaoga i totonu ole 10 aso). E i ai se uiga lelei ma alualu i luma i le fiafia o tagata faʻatau, o loʻo faʻaleleia pea e le matou faʻalapotopotoga a matou oloa lelei e faʻamalieina ai manaʻoga o tagata faʻatau ma sili atu ona taulaʻi i le saogalemu, faʻatuatuaina, faʻamatalaga o le siosiomaga, ma le faʻafouina o le Factory Supply China Convenient and D Quick Rt-Pforced work, faʻatauga Faʻatasi ma D Quick Rt-Ptepx. ia outou lagolago ma le loto atoa.Matou te faʻafeiloaʻi ma le faʻamaoni ia te oe e mautinoa e te tu i la matou upega tafaʻilagi ma kamupani ma meli mai ia i matou lau suʻesuʻega.
Sapalai FalegaosiSaina Taq DNA Polymerase, Qpcr, O loʻo i ai nei a matou fofo sili ona lelei ma faʻatauga faʻapitoa ma vaega faʻapitoa. Faatasi ai ma le atinaʻeina o la matou kamupani, e mafai ona matou tuʻuina atu i tagata faʻatau oloa sili ona lelei, lagolago faʻapitoa lelei, auaunaga atoatoa pe a uma ona faʻatau.

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