Multiplex qPCR Ikibazo cyambere
Injangwe No: HCB5051A
TaqMan multiplex qPCR Master Mix (Irangi Rishingiye) nigisubizo kibanziriza 2 × igihe nyacyo cyo kugereranya PCR amplification irangwa no kwiyumvamo ibintu byinshi kandi byihariye, bifite ubururu mubara, kandi bifite ingaruka zo kongeramo icyitegererezo.Iki gicuruzwa ni 2 × Kuvanga mbere-ivanze reagent ituma reaction zigera kuri enye za fluorescent PCR muburyo bumwe.Iki gicuruzwa kirimo uburyo bwa antibody bwahinduwe muburyo bwo gushyushya-gutangira Taq enzyme, bitezimbere cyane ibyiyumvo byiyongera kandi byihariye.Muri icyo gihe, iki gicuruzwa cyateje imbere cyane buffer-reaction, ishobora kunoza imikorere ya reaction kandi igateza imbere uburyo bwiza bwo gukwirakwiza inyandikorugero nke.Ibicuruzwa birashobora gukoreshwa muburyo bwa genotyping na multiplex isesengura ryinshi.
Ibisobanuro
| Intangiriro ishyushye | Byubatswe-bishyushye gutangira |
| Uburyo bwo kumenya | Kugaragaza Primer-probe |
| Uburyo bwa PCR | QPCR |
| Polymerase | Taq polymerase |
| Ubwoko bw'icyitegererezo | ADN |
Ububiko
Ibicuruzwa byoherejwe na barafu yumye kandi birashobora kubikwa kuri -25 ~ -15 ℃ kumyaka 2.
Amabwiriza
1. IgisubizoSisitemu
| Ibigize | Umubumbe (μL) | Kwibanda kwanyuma |
| 2 × TaqMan multiplex qPCR Master mix | 12.5 | 1 × |
| Kuvanga primer (10 μ mol / L) a | × | 0.1 - 0.5 μ mol / L. |
| Kuvanga ibibazo (10 μ mol / L)b | × | 50 - 250 nmol / L. |
| Irangi ryerekana irangi | 0.5 | 1 × |
| Inyandiko ya ADN / cDNA | 1-10 | - |
| ddH2O | kugeza kuri 25 | - |
Inyandiko:Kuvanga neza mbere yo gukoresha kugirango wirinde ibibyimba byinshi bitanyeganyega cyane.
a.Kwibanda kwa Primer: Primer Mix irimo ibice bibiri bya primers, mubisanzwe buri primer kumurongo wanyuma wa 0.2 μ mol / L kandi irashobora no guhinduka hagati ya 0.1 na 0.5 μ mol / L nkuko bikwiye.
b.Kwibanda kuri Probe: Probe Mix ikubiyemo iperereza ryinshi hamwe nibimenyetso bitandukanye bya fluorescence, kandi ubunini bwa buri probe burashobora guhinduka hagati ya 50 na 250 nmol / L ukurikije ibihe byihariye.
1.Rox dye reference: Ikoreshwa mugihe nyacyo PCR yongerera imbaraga nka Biosystems ikoreshwa kugirango ikosore ikosa ryibimenyetso bya fluorescence byakozwe hagati yamariba;iki gicuruzwa ntabwo kirimo Rox irangi.Cas # 10200 birasabwa niba bikenewe.
2.Inyandikorugero yerekana: qPCR irumva cyane, kandi birasabwa kugabanya inyandikorugero yo gukoresha.Niba inyandikorugero ari igisubizo cya cDNA, ingano yicyitegererezo ntigomba kurenza 1/10 cyubunini bwose.
3.Sisitemu yo kubyitwaramo: 25μL, 30μL cyangwa 50 μL irasabwa kwemeza imikorere nogusubiramo kwa gene amplification.
4.Gutegura sisitemu: Nyamuneka tegura intebe isukuye cyane, kandi ukoreshe inama hamwe nigituba kitagira ibisigisigi bya nuclease;birasabwa gukoresha inama hamwe na filteri ya karitsiye.Irinde kwanduza umusaraba no kwanduza aerosol.
2.Gahunda yo kubyitwaramo
| Intambwe | Ubushuhe. | Igihe | Amagare |
| Intangiriro-gutandukana | 95 ℃ | 5min | 1 |
| Gutandukana | 95 ℃ | 15sec | 45 |
| Kwiyongera / Kwagura | 60 ℃ | 30sec |
Inyandiko:
1.Annealing / Kwagura: Ubushyuhe nigihe birashobora guhinduka muburyo bukurikije primer Tm yagenewe.
2.Kugura ibimenyetso bya Fluorescence: Igihe cyo kubona ibimenyetso bya fluorescence gisabwa kubikoresho bitandukanye bya qPCR biratandukanye, nyamuneka shiraho ukurikije igihe ntarengwa.Igihe cyibikoresho byinshi bisanzwe gishyirwaho kuburyo bukurikira:
Amasegonda 20: Ikoreshwa ryibinyabuzima 7700, 7900HT, 7500 Byihuse
31 amasegonda: Ikoreshwa ryibinyabuzima 7300
32 amasegonda: Ikoreshwa ryibinyabuzima 7500
Inyandiko
Nyamuneka nyamuneka wambare PPE ikenewe, ikote rya laboratoire hamwe na gants, kugirango ubuzima bwawe n'umutekano wawe!
