page_banner

Ngwa Cell Direct RT qPCR — SYBR GREEN I

Nkọwa ngwa:

◮ Dị mfe ma dị irè: site na teknụzụ Cell Direct RT, enwere ike nweta ihe nlele RNA n'ime naanị nkeji 7.

Ọchịchọ nlele dị ntakịrị, ebe ọ bụ na enwere ike ịnwale mkpụrụ ndụ 10.

◮ Ntinye ego dị elu: ọ nwere ike ịchọpụta RNA ngwa ngwa n'ime mkpụrụ ndụ emebere na 384, 96, 24, 12, 6-well plates.

Nchicha DNA nwere ike wepu ngwa ngwa genomes ewepụtara, belata mmetụta na nsonaazụ nnwale na-esote.

Usoro RT na qPCR kachasị mma na-eme ka ntụgharị ntụgharị RT-PCR nzọụkwụ abụọ rụọ ọrụ nke ọma yana PCR akọwapụtara nke ọma, yana na-eguzogide ndị na-egbochi mmeghachi omume RT-qPCR.

foregene strength


Nkọwa ngwaahịa

Mkpado ngwaahịa

FAQ

Nkọwa

Ngwa a na-eji sistemu nchekwa lysis pụrụ iche nke nwere ike hapụ RNA ngwa ngwa site na nlele cell emebere maka mmeghachi omume RT-qPCR, si otú a na-ewepụ usoro ịdị ọcha nke RNA na-ewe ma na-arụsi ọrụ ike.Enwere ike nweta ndebiri RNA n'ime naanị nkeji 7.Nke 5×Direct RT Mix na 2×Direct qPCR Mix-SYBR reagents nke ngwa ahụ na-enye nwere ike nweta nsonaazụ PCR ozugbo na nke ọma.

5×Direct RT Mix na 2×Direct qPCR Mix-SYBR nwere nnabata inhibitor siri ike, enwere ike iji lysate nke ihe nlele ahụ dị ka ndebiri maka RT-qPCR ozugbo.Ngwa a nwere RNA pụrụ iche Foregene reverse transcriptase, yana Hot D-Taq DNA polymerase, dNTPs, MgCl.2, ihe nchekwa mmeghachi omume, PCR optimizer na stabilizer.

Nkọwapụta

200×20μl Rxns, 1000×20μl Rxns

Ngwa ngwa

Akụkụ I

Ihe nchekwa CL

Foregene Protease Plus II

Ihe nchekwa ST

Akụkụ II

Nchicha DNA

Ngwakọta 5 × Direct RT Mix

2× Direct qPCR Mix-SYBR

50× ROX ntụaka agbaji

ddH2O na-efu RNase

Ntuziaka

Atụmatụ & uru

Dị mfe ma dị irè: site na teknụzụ Cell Direct RT, enwere ike nweta ihe nlele RNA n'ime naanị nkeji 7.

■ Ọchịchọ nlele dị ntakịrị, ebe ọ bụ na enwere ike ịnwale mkpụrụ ndụ 10.

∎ Mgbakwunye dị elu: ọ nwere ike ịchọpụta RNA ngwa ngwa n'ime sel ndị emebere na 384, 96, 24, 12, 6-well plates.

∎ DNA Eraser nwere ike wepụ ngwa ngwa genomes ewepụtara, belata mmetụta dị na nsonaazụ nnwale na-esote.

∎ Sistemu RT na qPCR emeziri na-eme ka ntụgharị ntụgharị RT-PCR nzọụkwụ abụọ rụọ ọrụ nke ọma yana PCR akọwapụtara nke ọma, yana na-eguzogide ndị na-egbochi mmeghachi omume RT-qPCR.

Ngwa ngwa

Oke ngwa: mkpụrụ ndụ omenala.

- RNA wepụtara site na nlele lysis: naanị ọdabara na ndebiri RT-qPCR nke ngwa a.

- Enwere ike iji ngwa ahụ maka ebumnuche ndị a: nyocha okwu mkpụrụ ndụ ihe nketa, nkwenye nke mmetụta ịgbachitere mkpụrụ ndụ ihe nke siRNA, nyocha ọgwụ, wdg.

Eserese

Cell Direct RT qPCR diagram

Nchekwa na Shelf ndụ

A ga-echekwa akụkụ nke mbụ nke ngwa a na 4 ℃;Ekwesịrị ịchekwa akụkụ II na -20 ℃.

 Ekwesịrị ịchekwa Foregene Protease Plus II na 4℃, atụla oyi na -20 ℃.

 Reagent 2×Ekwesịrị ịchekwa Direct qPCR Mix-SYBR na -20n'ime ọchịchịrị;ọ bụrụ na a na-ejikarị ya eme ihe, a pụkwara ịchekwa ya na 4℃ maka nchekwa obere oge (jiri elu n'ime ụbọchị 10).


  • Nke gara aga:
  • Osote:

  • Real Time PCR primer ụkpụrụ imewe

    Na-aga n'ihu Primer na Reverse Primer

    Maka Real Time PCR, imewe primer dị ezigbo mkpa.Primers metụtara nkọwapụta na arụmọrụ nke nkwalite PCR, enwere ike hazie ya na ntụnyere ụkpụrụ ndị a:

    Ogologo oge mbụ: 18-30bp.

    Ọdịnaya GC: 40-60%.

    Uru Tm: Akụrụngwa imewe nke mbụ, dị ka Primer 5, nwere ike inye uru Tm nke primer.Ụkpụrụ Tm nke ndị na-ebuli elu na nke mgbada kwesịrị ịdị nso dịka o kwere mee.A nwekwara ike iji usoro mgbako Tm: Tm = 4 °C (G + C) + 2 Celsius C (A + T).Mgbe ị na-arụ PCR, a na-ahọrọ ọnọdụ okpomọkụ dị n'okpuru primer Tm uru nke 5 Celsius ka ọ bụrụ okpomọkụ na-ekpo ọkụ (mmụba kwekọrọ na okpomọkụ nke nkwụsịtụ nwere ike ime ka mmeghachi omume PCR pụta ìhè).

    Ngwaahịa mbụ na PCR:

    Nhazi primer PCR ogologo ngwaahịa nkwalite bụ ọkachamma 100-150bp.

    Ekwesịrị ịzere primers imewe na mpaghara nhazi nke abụọ nke ndebiri dị ka o kwere mee.

    Zenarị nhazi ntọala 2 ma ọ bụ karịa n'etiti nsọtụ 3 nke elu na mgbada ala.

    Primer 3′ ọdụ ọdụ enweghị ike ịnọ na 3 mgbakwunye G ma ọ bụ C.

    Ndị primers n'onwe ha enweghị ike ịnwe ihe mgbakwunye, ma ọ bụghị ya, a ga-emepụta nhazi ntutu isi, na-emetụta mmụba PCR.

    Ekwesịrị kesaa ATCG n'otu n'otu dịka o kwere mee n'usoro nke mbụ, yana 3′ ọdụ ọdụ kwesịrị izere ka T.

    Ihe mgbakwunye 1: Mkpọ mgbakwunye akụrụngwa Direct RT-qPCR Kit

    1.Cell Lysis Solution


    Cell Lysis Solution

    Ngwa ngwa

    (24-well lysis sistem / ọma)

    DRT-01011-A1

    DRT-01011-A2

    100 T

    500 T

    AkụkụI

    Ihe nchekwa CL

    20 ml

    100 ml

    Foregene Protease Plus II

    400 μl

    1 ml × 2

    Ihe nchekwa ST

    1 ml × 2

    10 ml

    AkụkụII

    Nchicha DNA

    400 μl

    1 ml × 2

    Ngwakọta 2.RT


    Ngwakọta RT

    Ngwa ngwa

    (Sistemụ mmeghachi omume 20 μl)

    DRT-01011-B1

    200 T

    Ngwakọta 5 × Direct RT Mix

    800 μl

    ddH na-efu RNase2O

    1.7 ml × 2

     

    Ngwakọta 3.qPCR


    Ngwakọta qPCR

    Ngwa ngwa

    (Sistemụ mmeghachi omume 20 μl)

    DRT-01011-C1

    DRT-01011-C2

    200 T

    1000 T

    2× Direct qPCR Mix-SYBR

    1 ml × 2

    1.7 ml × 6

    50× ROX ntụaka agbaji

    40 μl

    200 μl

    ddH na-efu RNase2O

    1.7 ml

    10 ml

    Dee ozi gị ebe a ziga anyị ya

    Ejikọtara yaNgwaahịa