(96-ọma) Ngwa ngwa ngwa ngwa ngwa ngwa RT-qPCR – SYBR Green I
Nkọwa
Nke(96-mma) QuickEasyTM Cell Direct RT-qPCR Kit-SYBR Green Ina-enyeusoro nchekwa lysis pụrụ icheto ngwa ngwa hapụ RNAsitere na cell omenalaihe nlele maka mmeghachi omume RT-qPCR, na-ewepụ oge na-ewe ma na-arụsi ọrụ ikeUsoro nchacha RNA, na-ewe naanị nkeji 7 iji nweta ndebiri RNA achọrọ.NkeNgwakọta 5 × Direct RT Mixna2× Direct qPCR Mix-SYBRnyere na igbe nwere ike ngwa ngwa nanweta ọnụọgụgụ ozugboNsonaazụ PCR.
5 × Direct RT Mix na 2 × Direct qPCR Mix-SYBR nwere nnabata inhibitor siri ike, ma nwee ike iji lysate nke ihe nlele a nwalere dị ka ndebiri maka ntụgharị ntụgharị nke ọma na nkwalite akọwapụtara.The reagent nwere Foregene pụrụ iche Reverse Transcriptase nwere nnukwu mmekọrịta maka RNA, yana Hot D-Taq DNA Polymerase, dNTPs, MgCl.2 , Nchikota mmeghachi omume, PCR optimizer and stabilizer , na enwere ike iji ya mee ihe na njikọ lysis iji chọpụta ihe nlele ngwa ngwa, dị mfe na n'ụzọ ziri ezi, na ọ nwere njirimara nke mmetụta dị elu, nkọwa siri ike na nkwụsi ike dị mma.
Ngwa ahụ bụ maka micro-system lysis nke 96-nke ọma sel, ma nwee ezigbo ịdị n'otu na nkwụsi ike;Ihe mejupụtara kit ahụ na-enye mmeghachi omume 96 lysis, mmeghachi omume ntụgharị 96 na mmeghachi omume 96 × 2 qPCR, nke nwere ike izute sel 96 nke ọma maka iji otu oge, na-ezere mmetọ nke imeghe ugboro ugboro, na-ajụ oyi na thawing nke reagents na mmebi nke arụmọrụ reagent.
Ngwa ngwa
| Ngwa mejupụtara ( 50 μSistemụ lysis / 20 μlRT usoro mmeghachi omume /20μSistemụ mmeghachi omume LqPCR) | DRT-03011 | kwuo | |
| 96T | |||
| AkụkụI | Ihe nchekwaCL | 5 ml | CellLysis |
| MbidoProtease Plus II | 100 μL | ||
| Ihe nchekwaST | 500 μL | ||
| Akụkụ II | DNANchicha | 100 μL | |
| Ngwakọta 5 × Direct RT Mix | 400 μL | RT | |
| 2 × Direct qPCR Mix-SYBR | 1 mL × 2 | qPCR | |
| 50× ROX ntụaka agbaji | 400µL | ||
| RNase- N'efuddH2 O | 1.7ml |
| |
| Mkwa afo | 1 ibe | 1 na-eje ozi | |
*: The lysis reagent DNA Eraser gụnyere na akụkụ II nke kit;Enwere ike zụta ihe mejupụtara Cell Lysis, RT na qPCR iche 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:
