5 × Neoscript yihuta RT-qPCR Premix wongeyeho-UNG

Injangwe No: HCB5142A

Neoscript Byihuta RT Premix-UNG (Probe qRT-PCR) ni imvururu ihamye cyane ya tube-probe ishingiye ku kuvanga ikwiranye n'intambwe imwe ihinduranya hamwe na PCR (qRT-PCR).Ifasha mbere yo kuvanga primers na probe kandi igahagarara neza nyuma yigihe kinini kibitse kubushyuhe buke.Icyitegererezo kizageragezwa gishobora kongerwaho muburyo butaziguye mugihe ukoresheje, nta miyoboro yinyongera ifungura / imiyoboro.Ibicuruzwa bitanga ibice, urugero nka polymerase ya ADN ishyushye, M-MLV, ubushyuhe-labile uracil ADN glycosylase (TS-UNG), RNase Inhibitor, MgCl₂, DNTPs (hamwe na dUTP mu mwanya wa dTTP), hamwe na stabilisateur.Hamwe na genetique yahinduwe byihuse amplification revers transcriptase na ADN polymerase, birashoboka kurangiza PCR amplification muminota 20-40.Iyi reagent ikoresha buffer idasanzwe kuri qPCR hamwe na enzymes zivanze za anti-inhibitory amplification enzyme na enzyme ya UNG.Kubwibyo, irashobora kubona amplifisione nziza ya gen igamije kandi ikarinda kwongerwaho ibinyoma biterwa na PCR isigaye hamwe n’umwanda wa aerosol.Iyi reagent irahujwe nibikoresho byinshi bya fluorescence yibikoresho bya PCR biva mubakora nka Applied Biosystems, Eppendorf, Bio-Rad na Roche.

Ibigize

1.25 × Neoscript yihuta RTase / UNG ivanze

2.5 × Neoscript yihuta ya RT Premix Buffer (dUTP)

Ububiko

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.

QRT-PCR Gutegura Sisitemu

1) a.Ubuso bwanyuma bwa primer mubusanzwe ni 0.2μM.Kubisubizo byiza, kwibanda kwa primer birashobora gutezimbere murwego rwa 0.2-1μM.Mubisanzwe, ubushakashatsi bwibanze bushobora gutezimbere murwego rwa 0.1-0.3μM.

2) b.Iyo ukoresheje uburyo bwihuse bwa PCR, kongera intumbero ya primers na probe bishobora kuvamo ibisubizo byiza bya amplification, kandi igipimo cyabo kigomba kuba cyiza bikurikije.

3) Ubwoko butandukanye bwintangarugero burimo ubwoko butandukanye nibirimo bya inhibitor na kopi ya gen igenewe.Ingano yicyitegererezo igomba gusuzumwa nuburyo nyabwo.Kora dilution yicyitegererezo hamwe namazi adafite nuclease cyangwa TE Buffer, nibiba ngombwa.

Igisubizo C.onditions

Kugenzura ubuziranenge

1.Kumenya imikorere: sensitivite, umwihariko no gusubiramo qPCR.

2.Nta gikorwa cya nuclease exogenous: nta exogenous endonuclease na exonuclease umwanda.

Inyandiko

1.Amplification igipimo cya ADN polymerase yihuta ntabwo iri munsi ya 1kb / 10s.Ibikoresho bitandukanye bya PCR bifite ubushyuhe butandukanye bwo gukonjesha no gukonjesha, uburyo bwo kugenzura ubushyuhe hamwe nubushyuhe bwumuriro, bityo rero gutezimbere primer / probe yibanze hamwe nuburyo bwo gukora bufatanije nibikoresho byawe byihuse PCR ni ngombwa.

2.Iki gicuruzwa gikora ibintu byinshi, kandi birakwiriye kwisuzumisha cyane.Uburyo butatu PCR uburyo busabwa kuri primers ifite ubushyuhe buke bwa annealing cyangwa kugirango hongerwe ibice birenga 200 bp.

3.Kubera ko amplicons zitandukanye zifite uburyo butandukanye bwo gukoresha dUTP hamwe nuburyo butandukanye kuri UNG, reagent igomba gutezimbere niba sensitivite yo kugabanuka igabanutse mugihe ukoresheje sisitemu ya UNG.Nyamuneka twandikire kugirango ubone inkunga ya tekiniki niba bikenewe.

4.Kugirango wirinde kwongera ibicuruzwa bya karryover PCR, ahantu hagenewe ubushakashatsi hamwe na pipette birasabwa kugirango byongerwe imbaraga.Koresha uturindantoki hanyuma uhindure kenshi kandi ntukingure umuyoboro wa PCR nyuma ya amplification.