Otu nzọụkwụ RT-qPCR Premix-UNG
Mpempe nwamba: HCB5145A
Multiplex One Step RT-qPCR Probe Kit (UDG Plus) bụ ngwa PCR ọnụọgụ ọnụọgụ dabere na RNA dị ka ndebiri.N'ime usoro nnwale ahụ, a na-eme ntụgharị ntụgharị na ọnụọgụ PCR n'otu tube ahụ, nke mere ka ọrụ nnwale dị mfe ma belata ohere nke mmetọ.N'ime ngwa a, eriri cDNA nke mbụ mebere nke ọma site na Reverse Transcriptase na-eguzogide okpomọkụ yana HotStart Tag DNA Polymerase kwalitere ya nke ọma.Ngwa ahụ na-enwekarị ihe nchekwa MP kachasị mma, ngwakọta enzymes, wdg. Ngwọta nchekwa ahụ nwere Mg2+na dNTP.Na mgbakwunye, a na-agbakwunye ihe ndị nwere ike igbochi mmetụ PCR na-abụghị nke akọwapụtara ma melite arụmọrụ mmụba nke ọtụtụ mmeghachi omume qPCR, nke nwere ike hụ na arụmọrụ mmụba na-aga n'ihu ruo ọtụtụ mmeghachi omume mmụba.Agbakwunyere usoro dUTP/UDG iji gbochie ihe ize ndụ nke mmetọ ikuku.
Ngwa
1. Nchekwa
2. Ngwakọta enzyme
Nkọwapụta
| Mmalite na-ekpo ọkụ | Nmalite ọkụ arụrụ arụ |
| Ụzọ nchọpụta | Nchọpụta nchọpụta mbụ |
| PCR usoro | Otu nzọụkwụ RT-qPCR |
| Polymerase | DNA polymerase |
| Ụdị ihe atụ | DNA |
Ọnọdụ Nchekwa
A na-ebufe ngwaahịa ahụ na ice akọrọ ma nwee ike ịchekwa ya na -25 ~ -15 ℃ maka 1 afọ.O kwesịrị izere ugboro ugboroifriizi-agbaze.A na-atụ aro ka ịchekwaa iche iche.
Ntuziaka
1.Mmeghachi omume Sistemu
| Ngwa | Olu (μL) | Ntụkwasị obi ikpeazụ |
| 2 × MP ihe nchekwa | 12.5 | 1× |
| Enzyme Mix | 1 | - |
| Ngwakọta Primer/Nchọpụta (2.5 μM) | 3 | 0.3 μM |
| Ụdị RNA | 1-10 | - |
| RNase H2O efu | ruo 25 | - |
ndetu:
Jide n'aka na ị ga-agwakọta nke ọma tupu ejiri ya, zere oke afụ nke ịma jijiji na-eme ihe ike kpatara.
a.Ntinye uche nke izizi: Ngwakọta Primer gụnyere multiplex primer, dabere na ọnọdụ kacha mma itinye uche n'etiti 0.l na 1.0μM.
b.Nleba anya nyocha: ngwakọta nyocha gụnyere multiplex probe labeling iche fluorescent otu, dabere na ọnọdụ ezigbo nyocha nyocha nwere ike n'etiti 0.05 na 0.5μM.
c.Ntughari ndebiri: qPCR na-enwe mmetụta nke ukwuu ma a na-atụ aro ka ị mebie ndebiri ahụ.Uru Ct njikwa dabara adaba n'etiti 20 na 35.
d.Nkwadebe usoro: Biko kwadebere na tebụl ọrụ dị ọcha dị ọcha, na pipettor na tube mmeghachi omume na-enweghị ihe fọdụrụ n'ime ihe nuclease;a na-atụ aro ka iji isi egbe nwere ihe nzacha.Zere mmetọ obe na mmetọ ikuku.
2. Ịnya igwe kachasị mmaProtocol
| okirikiri nzọụkwụ | Okpomọkụ | Oge | okirikiri |
| Ntugharị azụ azụ | 50 ℃a | Nkeji 20 | 1 |
| Mbido-denaturation | 95 ℃ | 5mins | 1 |
| Mmeghachi omume nkwalite | 95 ℃ | Nkeji 15 |
40-45 |
| 60 ℃ b | 30 sekọnd c |
ndetu:
a.Ntugharị ntụgharị: Okpomọkụ nwere ike họrọ 42°C ma ọ bụ 50°C.
b.Mmeghachi omume mmụba: A na-edozi ọnọdụ okpomọkụ dịka uru Tm nke primers emebere.
c.Nnweta mgbama fluorescence:Biko tọọ usoro nnwale dịka akwụkwọ ntuziaka akụrụngwa siri dị.
Ihe ndetu
Biko yiri PPE dị mkpa, dị ka uwe lab na uwe aka, iji hụ na ahụike na nchekwa gị.
