Lokho kunesimo sengqondo esihle nesiqhubekayo kokuthakaselayo kwamakhasimende, inhlangano yethu ithuthukisa ngokungaguquki ikhwalithi yemikhiqizo yethu ukuze inelise izimfuno zabathengi futhi igxile kakhulu ekuphepheni, ukwethembeka, ukucaciswa kwemvelo, kanye nokuqanjwa okusha kwe-Factory Supply China Convenient and Quick Rt-PCR Dspathtm 4X One-Step Multiplex Master Mix, singase sibe yizisebenzi zethu ezinolwazi olunzulu ngomsebenzi wethu.Siyakwamukela ngempela ukuthi uvakashele iwebhusayithi yethu kanye nenkampani futhi usithumele imibuzo yakho.
Lokho kunesimo sengqondo esihle nesiqhubekayo kokuthakaselayo kwamakhasimende, inhlangano yethu ihlale ithuthukisa ikhwalithi yemikhiqizo yethu ukuze yanelise izimfuno zabathengi futhi igxile kakhulu ekuphepheni, ekuthembekeni, ekucacisweni kwemvelo, nasekusunguleni izinto ezintsha.I-China Taq DNA Polymerase, Qpcr, Manje sinezixazululo ezingcono kakhulu nokuthengisa okunekhono kanye nethimba lobuchwepheshe.Ngokuthuthukiswa kwenkampani yethu, siyakwazi ukuhlinzeka amakhasimende ngemikhiqizo engcono kakhulu, ukusekelwa okuhle kwezobuchwepheshe, isevisi ephelele emva kokuthengisa.

Izincazelo

Le khithi isebenzisa isistimu ye-lysis buffer eyingqayizivele engakhulula ngokushesha i-RNA kumasampula eseli akhulisiwe ukuze kusabela i-RT-qPCR, ngaleyo ndlela iqede inqubo yokuhlanza i-RNA edla isikhathi nekhandlayo.Isifanekiso se-RNA singatholakala ngemizuzu engu-7 nje.I-5×Direct RT Mix kanye ne-2×Direct qPCR Mix-SYBR ama-reagents anikezwe ikhithi angathola ngokushesha nangempumelelo imiphumela ye-PCR yobuningi besikhathi sangempela.

I-5×Direct RT Mix kanye ne-2×Direct qPCR Mix-SYBR inokubekezelela okuqinile kwe-inhibitor, futhi i-lysate yamasampuli ingasetshenziswa njengesifanekiso se-RT-qPCR ngokuqondile.Le khithi iqukethe i-RNA high-affinity Foregene reverse transcriptase, kanye ne-Hot D-Taq DNA polymerase, dNTPs, MgCl₂, isigcinalwazi sokusabela, i-PCR optimizer nesiqinisi.

Imininingwane

200×20μl Rxns, 1000×20μl Rxns

Izingxenye zekhithi

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.

Umdwebo

Isitoreji kanye Nempilo yeshelufu

Ingxenye I yale kit kufanele igcinwe ku-4℃;Ingxenye II kufanele igcinwe ku -20 ℃.

I-Foregene Protease Plus II kufanele igcinwe ku-4℃, ingafrizi ku -20℃.

I-Reagent 2×Direct qPCR Mix-SYBR kufanele igcinwe ku -20℃ ebumnyameni;uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ ukuze igcinwe isikhathi esifushane (isetshenziswe zingakapheli izinsuku eziyi-10).Lokho kunesimo sengqondo esihle nesinenqubekelaphambili esithakazelweni samakhasimende, inhlangano yethu ithuthukisa ngokungaguquki ikhwalithi yemikhiqizo yethu ukuze yanelise izidingo zabathengi futhi igxile ekuphepheni, ekuthembekeni, ekucacisweni kwemvelo, nasekusungulweni okusha kwe-Factory Supply Chinat-RXTPCMR I-Multil Supply Chinat-RXTPCMR, I-Multil Supply Chinat-RXTPCPle I-Multi-Plus I-Quick-RX 4 Master abasebenzi abanolwazi oluyinkimbinkimbi bangase bakusekele ngenhliziyo yonke.Siyakwamukela ngempela ukuthi uvakashele iwebhusayithi yethu kanye nenkampani futhi usithumele imibuzo yakho.
Ukunikezwa KwemboniI-China Taq DNA Polymerase, Qpcr, Manje sinezixazululo ezingcono kakhulu nokuthengisa okunekhono kanye nethimba lobuchwepheshe.Ngokuthuthukiswa kwenkampani yethu, siyakwazi ukuhlinzeka amakhasimende ngemikhiqizo engcono kakhulu, ukusekelwa okuhle kwezobuchwepheshe, isevisi ephelele emva kokuthengisa.

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.

Isithasiselo 1:Iphakethe lengxenye ye-Cell Direct RT-qPCR Kit

1.Cell Lysis Solution