O le mauaina o le fa'amalieina o tagata fa'atau o le fa'amoemoe lea a le matou kamupani e le gata.O le a matou faia ni taumafaiga matagofie e gaosia ai oloa fou ma sili ona lelei, faʻamalieina ou manaʻoga faʻapitoa ma tuʻuina atu ia te oe le faʻatau muamua, faʻatau atu ma le maeʻa ona faʻatau atu auaunaga mo Top Quality China Universal Blue Sybr GreenQpcr Master Mixfa'atasi ai ma le Yellow Template Dilution Buffer, Matou te fa'amoemoe i le fa'aauauina o faiga fou, pulega fa'afouga, fa'afouga fa'apitoa ma pisinisi fou, tu'uina atu le ta'aloga atoa i tulaga lelei lautele, ma fa'aauau pea ona fa'aleleia atili le auaunaga maualuga.
O le mauaina o le fa'amalieina o tagata fa'atau o le fa'amoemoe lea a le matou kamupani e le gata.O le a matou faia ni taumafaiga matagofie e gaosia ai oloa fou ma sili ona lelei, faʻamalieina ou manaʻoga faʻapitoa ma tuʻuina atu ia te oe le muaʻi faʻatau atu, faʻatau atu ma le maeʻa ona faʻatau atu auaunaga moSaina Qpcr Premix, Qpcr Master Mix, O la matou upega tafa'ilagi fa'apitonu'u na fa'atupuina le silia ma le 50,000 fa'atauga fa'atau i tausaga ta'itasi ma manuia tele mo fa'atauga i luga ole laiga i Iapani.Matou te fiafia e maua se avanoa e fai pisinisi ma lau kamupani.Fa'atalitali i le mauaina o lau fe'au!

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 faʻaaogaina soo, e mafai foi ona teuina i le 4 ℃ mo le teuina mo taimi pupuu (faʻaaogaina i totonu ole 10 aso). O le mauaina o le faʻamalieina o tagata faʻatau o le faʻamoemoega a le matou kamupani e aunoa ma se mutaaga.O le a matou faia ni taumafaiga matagofie e gaosia ai oloa fou ma sili ona lelei, faʻamalieina ou manaʻoga faʻapitoa ma tuʻuina atu ia te oe le faʻatau muamua, faʻatau atu ma le maeʻa ona faʻatau atu auaunaga mo Top Quality China Universal Blue Sybr GreenQpcr Master Mixfa'atasi ai ma le Yellow Template Dilution Buffer, Matou te fa'amoemoe i le fa'aauauina o faiga fou, pulega fa'afouga, fa'afouga fa'apitoa ma pisinisi fou, tu'uina atu le ta'aloga atoa i tulaga lelei lautele, ma fa'aauau pea ona fa'aleleia atili le auaunaga maualuga.
Tulaga MaualugaSaina Qpcr Premix, Qpcr Master Mix, O la matou upega tafa'ilagi fa'apitonu'u na gaosia le silia ma le 50, 000 fa'atauga fa'atauga i tausaga ta'itasi ma manuia tele mo fa'atauga i luga ole laiga i Iapani.Matou te fiafia e maua se avanoa e fai pisinisi ma lau kamupani.Fa'atalitali i le mauaina o lau fe'au!

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