Foreasy Taq ADN Polymerase
Ibisobanuro:
◮Umwihariko wo hejuru: Enzyme ifite ibikorwa bishyushye-gutangira.
◮Kwiyongera byihuse: amasegonda 10 / kb.
◮Inyandikorugero ihindagurika cyane: irashobora gukoreshwa muguhindura neza GC agaciro gakomeye, ibintu bitandukanye-bigoye-kwagura inyandikorugero ya ADN.
◮Ubudahemuka bukomeye: Enzyme isanzwe ya Taq inshuro 6.
◮Ubushyuhe bukomeye bwumuriro: Irashobora gushyirwa kuri 37 ° C icyumweru kandi igakomeza ibikorwa birenga 90%.
Ibisobanuro
Foreasy Taq ADN Polymerase ni enzyme nshya ya Taq igaragara muri bagiteri ya Escherichia coli yubuhanga hakoreshejwe tekinoroji ya gene.Enzyme ubwayo ifite ibikorwa bishyushye-bitangira kandi birashobora gukoreshwa mubisanzwe PCR na qPCR;ifite ibikorwa 5 '→ 3' ibikorwa bya ADN polymerase na 5 '→ 3' ibikorwa bya exonuclease, ariko nta 3 '→ 5' ibikorwa bya exonuclease.
Ibigize ibikoresho
| Ibigize | IM-01011 | IM-01012 | IM-01013 |
| Foreasy Taq ADN Polymerase(5 U / μL) | 5000 U (1 mL) | 50 KU (10 mL) | 500 KU (100 mL) |
| 2 × Taf reaction ya Buffer | 25 mL × 5 | 250 mL × 5 | 500 mL × 25 |
Ibiranga ibyiza
- Umwihariko wo hejuru: Enzyme ifite ibikorwa runaka bishyushye-gutangira.
- Kwiyongera byihuse: amasegonda 10 / kb.
- Guhindura cyane inyandikorugero: irashobora gukoreshwa mugutezimbere neza GC Agaciro keza, ibintu bitandukanye-bigoye-kwagura inyandikorugero ya ADN.
- Ubudahemuka bukomeye: Enzyme isanzwe ya Taq inshuro 6.
- Ubushyuhe bukomeye bwumuriro: Irashobora gushyirwa kuri 37 ° C icyumweru kandi igakomeza ibikorwa birenga 90%
Porogaramu
Sisitemu zitandukanye za PCR / qPCR hamwe na sisitemu ya PCR
Kwiyongera kwa PCR ibice bya ADN
Ikimenyetso cya ADN
Urutonde rwa ADN
PCR A umurizo
U Ibisobanuro
1U: Ingano ya enzyme isabwa kugirango yinjize nmol 10 ya deoxynucleotide mu kintu cya aside-idashobora gukoreshwa ukoresheje intanga ngabo ya salmon ikora nka template / primer muminota 30 kuri 74 ° C.
Imiterere
| Ubushyuhe | Igihe | Ukuzenguruka |
| 37 ° C. | 5min | 1 |
| 94 ° C. | 5min | 1 |
| 94 ° C. | Amasegonda 10 | 35 |
| 60 ° C. | Amasegonda 10 | |
| 72 ° C. | 20 amasegonda / kb | |
| 72 ° C. | 2min | 1 |
Ububiko
-20 ± 5 ° C kumyaka 2 cyangwa kuri -80 ° C kubikwa igihe kirekire.
Nta kimenyetso cyo kongera imbaraga
1.Tak ADN Polymerase ya Taq mubikoresho itakaza ibikorwa byayo kubera kubika nabi cyangwa kurangira ibikoresho.
Icyifuzo: Emeza uburyo bwo kubika ibikoresho;ongera wongere umubare ukwiye wa Taq DNA Polymerase muri sisitemu ya PCR cyangwa ugure ibikoresho bishya bya PCR Igihe cyakorewe ubushakashatsi bujyanye.
2.Hariho byinshi bibuza Taq DNA Polymerase mubishusho bya ADN.
Igitekerezo: Ongera usubize inyandikorugero cyangwa ugabanye urugero rwicyitegererezo cyakoreshejwe.
3.Ibitekerezo bya Mg2 + ntibikwiye.
Icyifuzo: Mg2 + kwibanda kuri2 × Imvange ya PCR nyayo dutanga ni 3.5mM.Ariko, kuri bimwe bidasanzwe primers hamwe na templates, kwibanda kwa Mg2 + birashobora kuba hejuru.Kubwibyo, urashobora kongeramo MgCl2 kugirango uhindure ibitekerezo bya Mg2 +.Birasabwa kongera Mg2 + 0.5mM buri gihe kugirango optimiz.
4.Ibihe byo kongera PCR ntabwo bikwiye, kandi primer ikurikirana cyangwa kwibandaho ntibikwiye.
Igitekerezo: kwemeza ukuri kwa primer bikurikiranye kandi primer ntabwo yateshejwe agaciro;niba ibimenyetso bya amplification atari byiza, gerageza kugabanya ubushyuhe bwa annealing hanyuma uhindure primer yibanze neza.
5.Ubunini bwicyitegererezo ni gito cyane cyangwa byinshi.
Icyifuzo: Kora icyitegererezo cyerekana umurongo ugereranya, hanyuma uhitemo icyitegererezo cyibanze hamwe nibyiza bya PCR kubigeragezo nyabyo PCR.
NTC ifite agaciro ka fluorescence cyane
1.Ubwandure bukabije buterwa mugihe cyo gukora.
Icyifuzo: Simbuza reagent nshya kubihe nyabyo PCR igerageza.
2.Kwanduza kwabaye mugihe cyo gutegura sisitemu ya reaction ya PCR.
Icyifuzo: Fata ingamba zikenewe zo kurinda mugihe cyo gukora, nka: kwambara uturindantoki twa latex, ukoresheje umuyoboro wa pipette ufite akayunguruzo, nibindi.
3.Ibibanza byangiritse, kandi gutesha agaciro primers bizatera amplification idasanzwe.
Igitekerezo: Koresha SDS-PAGE electrophoreis kugirango umenye niba primers yangiritse, hanyuma uyisimbuze primers nshya kubushakashatsi bwa Real Time PCR.
Primer dimer cyangwa idasanzwe idasanzwe
1.Ibitekerezo bya Mg2 + ntibikwiye.
Icyifuzo: Mg2 + kwibanda kuri 2 × Real PCR EasyTM ivanze dutanga ni 3.5 mM.Ariko, kuri bimwe bidasanzwe primers hamwe na templates, kwibanda kwa Mg2 + birashobora kuba hejuru.Kubwibyo, urashobora kongeramo MgCl2 kugirango uhindure ibitekerezo bya Mg2 +.Birasabwa kongera Mg2 + 0.5mM buri gihe kugirango optimiz.
2.Ubushyuhe bwa PCR buri hasi cyane.
Igitekerezo: Ongera ubushyuhe bwa PCR kuri 1 ℃ cyangwa 2 ℃ buri gihe.
3.Ibicuruzwa bya PCR ni birebire cyane.
Icyifuzo: Uburebure bwibicuruzwa nyabyo PCR bigomba kuba hagati ya 100-150bp, ntibirenze 500bp.
4.Ibibanza byangiritse, kandi gutesha agaciro primers bizaganisha ku kugaragara kwa amplification yihariye.
Igitekerezo: Koresha SDS-PAGE electrophoreis kugirango umenye niba primers yangiritse, hanyuma uyisimbuze primers nshya kubushakashatsi bwa Real Time PCR.
5. Sisitemu ya PCR idakwiye, cyangwa sisitemu ni nto cyane.
Icyifuzo: Sisitemu ya reaction ya PCR ni nto cyane bizatera kumenya neza kugabanuka.Nibyiza gukoresha sisitemu ya reaction isabwa nigikoresho cya PCR kugirango wongere ukore igeragezwa ryigihe PCR.
Gusubiramo nabi kwindangagaciro
1.Igikoresho kirimo gukora nabi.
Igitekerezo: Hashobora kubaho amakosa hagati ya buri mwobo wa PCR wigikoresho, bikavamo imyororokere mibi mugihe cyo gucunga ubushyuhe cyangwa gutahura.Nyamuneka reba ukurikije amabwiriza y'igikoresho kijyanye.
2.Icyitegererezo cyera ntabwo ari cyiza.
Icyifuzo: Ingero zanduye zizaganisha ku myororokere mibi yubushakashatsi, burimo ubuziranenge bwicyitegererezo na primers.Nibyiza gusubiramo inyandikorugero, kandi primers zezwa neza na SDS-PAGE.
3.Itegurwa rya sisitemu ya PCR nigihe cyo kubika ni kirekire cyane.
Igitekerezo: Koresha sisitemu nyayo PCR kubushakashatsi bwa PCR ako kanya nyuma yo kwitegura, kandi ntugasige kuruhande igihe kirekire.
4.Ibihe byo kongera PCR ntabwo bikwiye, kandi primer ikurikirana cyangwa kwibandaho ntibikwiye.
Igitekerezo: kwemeza ukuri kwa primer bikurikiranye kandi primer ntabwo yateshejwe agaciro;niba ibimenyetso bya amplification atari byiza, gerageza kugabanya ubushyuhe bwa annealing hanyuma uhindure primer yibanze neza.
5. Sisitemu ya PCR idakwiye, cyangwa sisitemu ni nto cyane.
Icyifuzo: Sisitemu ya reaction ya PCR ni nto cyane bizatera kumenya neza kugabanuka.Nibyiza gukoresha sisitemu ya reaction isabwa nigikoresho cya PCR kugirango wongere ukore igeragezwa ryigihe PCR.
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