Ngwa Cell Direct RT qPCR—Taqman Direct Cell Lysis Cell njikere otu nzọụkwụ qRT-PCR ngwa nyocha ngwa
Nkọwa
Ngwaahịa a na-eji sistemụ nchekwa lysis pụrụ iche iji wepụta RNA ngwa ngwa site na ihe nlele cell emebere maka mmeghachi omume RT-qPCR, na-ewepụ usoro ịdị ọcha nke RNA na-eri oge na nke siri ike, yana naanị nkeji 7 iji nweta ndebiri RNA achọrọ, yana 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman nyere site na ngwa ahụ nwere ike nweta ngwa ngwa PCR na ezigbo oge.
5 × Direct RT Mix na 2 × Direct qPCR Mix-Taqman nwere nnabata inhibitor siri ike ma nwee ike ịrụ ọrụ ntụgharị nke ọma na nkwalite akọwapụtara site na iji lysate nke ihe atụ a ga-atụle dị ka ndebiri.Reagent nwere Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl.2, Reaction Buffer, PCR Optimizer na Stabilizer, nke enwere ike iji ya na lysis buffer iji chọpụta ngwa ngwa ngwa ngwa, ma nwee njirimara nke mmetụta dị elu, nkọwa na nkwụsi ike.
Nkọwapụta
200×20μl Rxns, 1000×20μl Rxns
Ngwa ngwa
| Ọ dị mfeTMCell Direct RT-qPCR Kit-Taqman | ||||
| Ngwa ngwa Sistemụ mmeghachi omume 20μl qPCR | DRT-01021 | DRT-01022 | Rịba ama | |
| 200 T | 1000 T | |||
|
Akụkụ I | Ihe nchekwa CL | 4 ml | 20 ml | Cell Lysis |
| Foregene Protease Plus II | 80 μl | 400 μl | ||
| Ihe nchekwa ST | 400 μl | 1 ml × 2 | ||
|
Akụkụ II | Nchicha DNA | 80 μl | 400 μl | |
| Ngwakọta 5 × Direct RT * | 160 μl | 800 μl | RT | |
| 2 × Direct qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
| 20×ROX ntụaka agbaji | 40 μl | 200 μl | ||
| ddH na-efu RNase2O | 1.7 ml | 10 ml | ||
| Akwụkwọ ntuziaka | 1 ibe | 1 ibe | ||
*:Cell Lysis, 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman nwere ike zụta iche.
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 emezigharịrị na-eme ka ntụgharị ntụgharị RT-PCR nzọụkwụ abụọ rụọ ọrụ nke ọma yana PCR akọwapụtara nke ọma, ma 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.
Nchekwa na Shelf ndụ
Ekwesịrị ịchekwa akụkụ I 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-Taqman na -20℃n'ime ọchịchịrị;ọ bụrụ na-eji ya ugboro ugboro, a pụkwara ịchekwa ya na 4℃ maka nchekwa obere oge (jiri elu n'ime ụbọchị 10).
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 okpomọkụ dị n'okpuru uru Tm primer nke 5 Celsius dị ka okpomọkụ na-ekpo ọkụ (mmụba kwekọrọ na okpomọkụ na-ekpo ọkụ nwere ike ịbawanye nkọwa nke mmeghachi omume PCR).
- Ngwaahịa mbụ na PCR:
- Design primer PCR amplification ogologo ngwaahịa 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 ala ala.
- Primer 3′ terminal base enweghị ike ịnọ ya 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 usoro ntutu isi, na-emetụta mmụba PCR.
- Ekwesịrị kesaa ATCG n'otu n'otu dịka o kwere mee n'usoro nke mbụ, yana ebe njedebe 3′ kwesịrị izere ka T.
Ihe mgbakwunye1:Cebe DirectRT-qPCR Ngwakọta ihet mgbakwunye mkpọ
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 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 qPCR | ||
| Ngwa ngwa (Sistemụ mmeghachi omume 20 μl) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2 × Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
| 20× ROX ntụaka agbaji | 40 μl | 200 μl |
| ddH na-efu RNase2O | 1.7 ml | 10 ml |
Akwụkwọ ntuziaka:
