Ngwa Cell Direct RT qPCR—SYBR GREEN M na-eduzi Cell Lysis Cell njikere otu nzọụkwụ qRT-PCR ngwa
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 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.
Eserese
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 -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 mgbakwunye 1: Mkpọ mgbakwunye akụrụngwa Direct RT-qPCR Kit
1.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
|
| |
| 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 |
|
| ||
| 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 |
Akwụkwọ ntuziaka:
