2 × Sensi Yibanze-UNG (Probe qPCR)
Injangwe No: HCB5151A
SensiDirect Premix-UNG (Probe qPCR) yagenewe gukora PCR biturutse ku ngero zidakuweho ADN cyangwa ngo itegure icyitegererezo.Iyi reagent igizwe na polymerase ya ADN ishyushye, uracil ADN glycosylase (UNG), Inhibitor ya RNase, MgCl2, DNTPs (hamwe na dUTP mu mwanya wa dTTP), hamwe na stabilisateur, kuri PCR yuzuye (qPCR).Iyi reagent yerekana kwihanganira cyane, bityo irashobora gukoreshwa muburyo bwo kumenya ingero nka mu muhogo, amacandwe, kurwanya amaraso yose, plasma, na serumu nta gukuramo ADN.Reagent ikoresha buffer yihariye kuri qPCR hamwe na enzymes zivanze za anti-inhibitory ADN polymerase na enzyme ya UNG.Kubwibyo, irashobora kubona amplifisione nziza ya gen igenewe mubitegererezo birimo inhibitor kandi ikabuza amplifisation yibinyoma iterwa na PCR isigara hamwe na aerosol.Iyi reagent irahujwe nibikoresho byinshi bya fluorescent PCR yibikoresho, nka Biosystems ikoreshwa, Eppendorf, Bio-Rad, Roche nibindi.
Ibigize
1. 50 × Enzyme ya SensiDirect / UNG ivanze
2. 2 × SensiDirect Premix Buffer (dUTP)
Imiterere yo kubika
Ibigize byose bigomba kubikwa kuri -20 ℃ kubikwa igihe kirekire na 4 ℃ kugeza kumezi 3.Nyamuneka vanga neza nyuma yo gusya na centrifuge mbere yo gukoresha.Irinde gukonjesha kenshi.
Umukino wo gusiganwa ku magare
| Intambwe | Ubushyuhe | Igihe | Ukuzenguruka |
| Gusya | 50 ℃ | 2min | 1 |
| Gukora polymerase | 95 ℃ | 1-5min | 1 |
| Kwanga | 95 ℃ | 10-20 | 40-50 |
| Kwiyongera / Kwagura | 56-64 ℃ | 20-60s |
Amabwiriza yo kuvoma
| Reagent | Umubumbe kuri reaction | Ingano kuri buri reaction | Kwibanda kwanyuma |
| 2 × SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1 × |
| 50 × Ensime ya SensiDirect / UNG ivanze | 0.5µL | 1µL | 1 × |
| 25 × Kuvanga Primer-Probe1, 2 | 1µL | 2µL | 1 × |
| Icyitegererezo3, 4 | - | - | - |
| ddH2O | - | - | - |
| Ingano yose | 25 μL | 50 μL | - |
1. Kwibanda kwanyuma kwa primer mubisanzwe ni 0.2μM.Kubisubizo byiza, kwibanda kwa primer birashobora gutezimbere murwego rwa 0.2-1μM.
2. Mubisanzwe, ubushakashatsi bwibanze bushobora gutezimbere murwego rwa 0.1-0.3μM.Ubwiza bwiza bwa theprobe bujyanye nigihe nyacyo igikoresho cyo kongera PCR, ubwoko bwa probe, nubwoko bwa fluorescent labels ibintu.Nyamuneka reba igitabo gikubiyemo ibikoresho cyangwa ibisabwa byihariye bya buri kintu cya fluorescent.
3. Ubwoko butandukanye bwintangarugero burimo ubwoko butandukanye nibirimo bya inhibitor hamwe na kopi ya gen igenewe.Ingano yicyitegererezo igomba gusuzumwa nuburyo nyabwo.Kora dilution yicyitegererezo wongeyeho nuclease idafite amazi cyangwa TE Buffer, nibiba ngombwa.
4. Ingano isabwa yingero zitandukanye:
| Icyitegererezo | Umubumbe umwe 50 μL reaction | Ntarengwa Ikigereranyo |
| Amaraso yuzuye | 2.5 μL | 5% |
| Plasma | 15 μL | 30% |
| Serumu | 10 μL | 20% |
| Umuhogo | 10 μL | 20% |
| Amacandwe | 10 μL | 20% |
Kugenzura ubuziranenge
1. Kumenya imikorere: sensitivite, umwihariko no gusubiramo qPCR.
2. Nta gikorwa cya nuclease exogenous: nta exogenous endonuclease na exonuclease umwanda.
Inyandiko y'ibicuruzwa
1. Iki gicuruzwa gikoresha ubwoko bushya bwa polymerase ya ADN ishyushye, ishobora gukora muminota 1-5.Kuberako reaction ya reaction yayo imaze kuba nziza, irakwiriye cyane kuri PCR inshuro ebyiri cyangwa nyinshi za PCR ikoresheje uburyo bwa probe.
2. Niba agaciro ka Rn kongererwa PCR ari hasi cyane cyangwa amplification biragaragara ko yabujijwe, kugabanya urugero rwicyitegererezo, kongera urugero rwibisubizo cyangwa kugabanuka kwicyitegererezo bishobora kunoza ibisubizo.
3. Ikusanyirizo ryamaraso, amacandwe, inkari, umuhogo, nibindi bigomba gukurikiza ibisabwa nubuvuzi, kandi icyitegererezo gishya gishobora gukoreshwa kugirango wirinde kwangirika kwa aside nucleique.
4. Kubera ko amplicons zitandukanye zifite uburyo butandukanye bwo gukoresha kuri dUTP no kumva neza UNG, reagent igomba gutezimbere niba sensitivite yo kugabanuka igabanutse mugihe ukoresheje sisitemu ya UNG.Nyamuneka twandikire kugirango ubone inkunga ya tekiniki niba bikenewe.
5. Kugira ngo wirinde kwongera ibicuruzwa bya karryover PCR hagati yintambwe imwe, ahantu hagenewe ubushakashatsi hamwe na pipette birakenewe kugirango byongerwe imbaraga.Koresha na gants hanyuma uhindure kenshi kandi ntukingure reaction ya reaction nyuma ya PCR.
