5× Neoscript ngwa ngwa RT-qPCR Premix gbakwunyere-UNG
Mpempe nwamba: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) bụ ngwakọta dabere na otu tube nke dabara adaba maka ntụgharị ntụgharị otu nzọụkwụ na PCR (qRT-PCR).Ọ na-akwado tupu ịgwakọta primers na nyocha ma kwụsie ike mgbe nchekwa ogologo oge gasịrị na obere okpomọkụ.Enwere ike ịgbakwunye ihe nlele a ga-anwale ozugbo mgbe ị na-eji ya, na-enweghị ọrụ oghere tube / ọkpọkọ ọzọ.Ngwaahịa a na-enye akụrụngwa, dịka DNA polymerase na-ekpo ọkụ, M-MLV, uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl.2, dNTPs (ya na dUTP kama dTTP), na stabilizers.Site na mgbanwe mgbanwe mkpụrụ ndụ ihe nketa ngwa ngwa reverse transcriptase na DNA polymerase, ọ ga-ekwe omume ịmechaa nkwalite PCR n'ime nkeji 20-40.Nke a reagent na-eji ihe nchekwa pụrụ iche maka qPCR yana enzymes agwakọta nke enzyme mgbochi mgbochi mgbochi yana UNG enzyme.Ya mere, ọ nwere ike nweta nkwalite dị mma nke mkpụrụ ndụ ihe nketa lekwasịrị anya ma gbochie mmụba ụgha nke PCR residual na aerosol kpatara.Nke a reagent dakọtara na ọtụtụ fluorescence quantitative PCR ngwa sitere na ndị nrụpụta dị ka Applied Biosystems, Eppendorf, Bio-Rad na Roche.
Akụkụ
1.25× Neoscript ngwa ngwa RTase/UNG Mix
2.5 × Neoscript ngwa ngwa RT Premix Buffer (dUTP)
Ọnọdụ Nchekwa
All components kwesịrị idobe na -20 ℃ maka nchekwa ogologo oge yana 4 ℃ ruo ọnwa 3.Biko gwakọta nke ọma mgbe ịchasịrị na centrifuge tupu i jiri ya.Zere na-agbaze oyi mgbe niile.
Nkwadebe Sistemu mmeghachi omume qRT-PCR
| Ngwa | 25μLSistemu | 50μLSistemu | Ntụkwasị obi ikpeazụ |
| 5 × Neoscript ngwa ngwa RT Premix Buffer (dUTP) | 5μl | 10μL | 1× |
| 25× Neoscript ngwa ngwa RTase/UNG Mix | 1μl | 2μL | 1× |
| Ngwakọta 25 × Primer-Probe Mixa | 1μl | 2μL | 1× |
| Ụdị RNAb | - | - | - |
| ddH2O | Ihe dị ka 25 μl | Ihe ruru 50 μl | - |
1) a.Ntụle ikpeazụ nke primer na-abụkarị 0.2μM.Maka nsonaazụ kacha mma, enwere ike ịkwalite ntinye nke primer n'ime oke nke 0.2-1μM.N'ozuzu, enwere ike ịkwalite ntinye nyocha n'ime oke nke 0.1-0.3μM.
2) b. Mgbe ị na-eji usoro PCR ngwa ngwa, ịba ụba nke primers na nyocha nwere ike ime ka nsonaazụ mmụba dị mma, na nha ha kwesịrị ka ọ dịkwuo mma.
3) Ụdị ihe atụ dị iche iche nwere ụdị dị iche iche na ọdịnaya nke inhibitor na nọmba nke mkpụrụ ndụ ihe mgbaru ọsọ.Ekwesịrị ịtụle olu nlele ahụ site na ọnọdụ n'ezie.Mee dilution nke ihe nlele na mmiri na-enweghị nuclease ma ọ bụ TE Buffer, ọ bụrụ na ọ dị mkpa.
Mmeghachi omume Cmmalite
| Usoro PCR oge niile | Usoro PCR ngwa ngwa | ||||||
| Usoro | Okpomọkụ | Oge | okirikiri | Usoro | Okpomọkụ | Oge | okirikiri |
| Ntugharị ntụgharị | 50 ℃ | 10-20 nkeji | 1 | Ntugharị ntụgharị | 50 ℃ | 5mins | 1 |
| Polymerase Nkwalite | 95 ℃ | 1-5 nkeji | 1 | Polymerase Nkwalite | 95 ℃ | 30s | 1 |
| Denaturation | 95 ℃ | 10-20s | 40-50 | Denaturation | 95 ℃ | 1-3s | 40-50 |
| Na-ewe iwe na Mgbatị | 56-64 ℃ | Afọ 20-60 | Na-ewe iwe na Mgbatị | 56-64 ℃ | 3-20s | ||
Njikwa ogo
1.Nchọpụta ọrụ: uche, nkọwapụta na nkwughachi nke qPCR.
2.Ọ dịghị ọrụ nuclease exogenous: ọ dịghị exogenous endonuclease na exonuclease mmetọ.
Ihe ndetu
1.Ọnụego mmụba nke DNA polymerase ngwa ngwa erughị 1kb/10s.Ngwa PCR dị iche iche nwere ọsọ kpo oku na oyi dị iche iche, ụdị njikwa okpomọkụ na nrụpụta ọkụ, yabụ njikarịcha nke ntinye uche / nyocha gị yana usoro ịgba ọsọ yana ngwa ngwa PCR gị dị mkpa.
2.Ngwaahịa a na-arụ ọrụ dị ukwuu, yana ọ dabara maka nchọpụta molekul nwere mmetụta dị elu.A na-atụ aro usoro PCR nzọụkwụ atọ maka primers nwere obere okpomọkụ na-ekpo ọkụ ma ọ bụ maka mmụba nke iberibe ogologo karịa 200 bp.
3.Ebe ọ bụ na amplicon dị iche iche nwere ọrụ dị iche iche nke dUTP na mmetụta dị iche iche na UNG, ekwesịrị ịkwalite reagents ma ọ bụrụ na nchọpụta nchọpụta na-ebelata mgbe ị na-eji usoro UNG.Biko kpọtụrụ anyị maka nkwado teknụzụ ma ọ bụrụ na ọ dị mkpa.
4.Iji zere nkwalite ngwaahịa PCR na-ebu, a na-achọ ebe nnwale na pipette maka nkwalite.Jiri uwe aka na-arụ ọrụ ma na-agbanwe ugboro ugboro ma emeghela PCR tube mgbe nkwalite.
