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I-Cell Direct RT qPCR Kit—SYBR GREEN I

Incazelo yekhithi:

◮Ilula futhi iyasebenza: Ngobuchwepheshe be-Cell Direct RT, amasampula e-RNA angatholwa ngemizuzu engu-7 nje.

Isidingo sesampula sincane, siphansi njengamaseli ayi-10 angahlolwa.

◮ Ukusebenza okuphezulu: ingakwazi ukubona ngokushesha i-RNA kumaseli akhuliswe kumapuleti angu-384, 96, 24, 12, 6-wemithombo.

I-DNA Eraser ingasusa ngokushesha ama-genome akhululiwe, inciphise kakhulu umthelela emiphumeleni yokuhlola elandelayo.

I-RT ethuthukisiwe nesistimu ye-qPCR yenza ukuloba okuhlanekezela okuzinyathelo ezimbili kwe-RT-PCR kusebenze kahle kakhulu futhi i-PCR icace kakhudlwana, futhi imelane kakhulu nama-RT-qPCR reaction inhibitors.

foregene strength


Imininingwane Yomkhiqizo

Omaka bomkhiqizo

FAQ

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 eyi-7 nje.I-5×I-Direct RT Mix kanye no-2×Ama-reagents aqondile we-qPCR Mix-SYBR ahlinzekwa yikhithi angathola ngokushesha nangempumelelo imiphumela ye-PCR yobuningi besikhathi sangempela.

5×I-Direct RT Mix kanye no-2×I-Direct qPCR Mix-SYBR inokubekezelela okuqinile kwe-inhibitor, futhi i-lysate yamasampuli ingasetshenziswa njengesifanekiso se-RT-qPCR ngokuqondile.Le kit 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 rxn

Izingxenye zekhithi

Ingxenye I

Isivimbeli CL

I-Foregene Protease Plus II

Isilondolozi se-ST

Ingxenye II

I-DNA Eraser

5× Imiksi ye-RT eqondile

2× Direct qPCR Mix-SYBR

I-50 × ROX Reference Dye

I-RNase-Free ddH2O

Iziyalezo

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.

■ Ukusebenza 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 izinyathelo ezimbili ze-RT-PCR zibhale ngokuhlehlayo zisebenze kahle futhi i-PCR icace kakhulu, futhi imelane kakhulu nama-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

Cell Direct RT qPCR diagram

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℃, ungafrizi ku -20℃.

 I-Reagent 2×I-QPCR Mix-SYBR eqondile kufanele igcinwe ku -20ebumnyameni;uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ ukuze ugcine isikhathi esifushane (sebenzisa phakathi kwezinsuku eziyi-10).


  • Okwedlule:
  • Olandelayo:

  • 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 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′ weziqalo 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


    Cell Lysis Solution

    Izingxenye zekhithi

    (Isistimu ye-lysis ye-24 / kahle)

    I-DRT-01011-A1

    I-DRT-01011-A2

    100 T

    500 T

    IngxenyeI

    Isivimbeli CL

    20 ml

    100 ml

    I-Foregene Protease Plus II

    400 μl

    1 ml × 2

    Isilondolozi se-ST

    1 ml × 2

    10 ml

    IngxenyeII

    I-DNA Eraser

    400 μl

    1 ml × 2

    2.RT Imiksi


    I-RT Mix

    Izingxenye zekhithi

    (Isistimu yokusabela engu-20 μl)

    I-DRT-01011-B1

    200 T

    5× Imiksi ye-RT eqondile

    800 μl

    I-ddH yamahhala ye-RNase2O

    1.7 ml × 2

     

    3.qPCR Mix


    I-qPCR Mix

    Izingxenye zekhithi

    (Isistimu yokusabela engu-20 μl)

    I-DRT-01011-C1

    I-DRT-01011-C2

    200 T

    1000 T

    2× Direct qPCR Mix-SYBR

    1 ml × 2

    1.7 ml × 6

    I-50 × ROX Reference Dye

    awu 40l

    200 μl

    I-ddH yamahhala ye-RNase2O

    1.7 ml

    10 ml

    Bhala umlayezo wakho lapha futhi usithumelele wona

    OkuhlobeneImikhiqizo