Robustart Taq ADN Polymerase

Robustart Taq ADN Polymerase nintangiriro ishyushye ya polymerase ya ADN.Iki gicuruzwa ntigishobora gusa kubuza gusa uburyo budasanzwe bwatewe na annealing idasanzwe ya primers cyangwa guteranya primer mugikorwa cyo gutegura sisitemu ya PCR no kuyongera.Kubwibyo, ifite umwihariko mwiza kandi ifite akamaro kanini mugukwirakwiza inyandikorugero ntoya, kandi irakwiriye kugwiza PCR kwinshi.Byongeye kandi, iki gicuruzwa gifite akamaro gakomeye cyane, kandi ibisubizo bihamye bya amplification birashobora kuboneka muburyo butandukanye bwa PCR.

Ibigize

1.5 U / μL Robustart Taq ADN polymerase

2.10 × PCR Buffer II (Mg² + ubuntu) (bidashoboka)

3.25 mM MgCl₂(bidashoboka)

* 10 × PCR Buffer II (Mg² + ubuntu) ntabwo irimo dNTP na Mg² +, nyamuneka ongeraho DNTPs na MgCl₂mugihe utegura sisitemu yo kwitwara.

Gusabwa

1.Kwiyongera vuba.

2.Kwiyongera kwinshi.

3.Kwiyongera kwamaraso, swabs, nizindi ngero.

4.Indwara z'ubuhumekero.

Imiterere y'Ububiko

-20 ° C kubikwa igihe kirekire, bigomba kuvangwa neza mbere yo kubikoresha, irinde gukonjesha kenshi.

* Niba imvura iguye nyuma yo gukonjesha, nibisanzwe;birasabwa kuringaniza ubushyuhe bwicyumba mbere yo kuvanga no gukoresha.

Igisobanuro

Igice kimwe gikora (U) gisobanurwa nkubunini bwa enzyme yinjizamo nmol 10 ya deoxyribonucleotide mubikoresho bya aside-idashonga kuri 74 ° C kuri 30mins ukoresheje intanga ngabo ya salmon ikora nka template / primer.

Kugenzura ubuziranenge

1.SDS-PAGE isuku ya electrophoreque irenga 98%.

2.Amplification sensitivite, icyiciro-kuri-kugenzura, gutuza.

3.Ntabwo ibikorwa bya nuclease bidasanzwe, nta endogenuclease ya exogenous cyangwa exonuclease yanduye

Amabwiriza

Gushiraho

Inyandiko:

1) a.Buffer ntabwo irimo DNTP na Mg² +, nyamuneka ongeraho DNTPs na MgCl₂mugihe utegura sisitemu yo kwitwara.

2) b.Niba ikoreshwa kuri qPCR / qRT-PCR, fluorescent probe igomba kongerwaho sisitemu ya reaction.Mubisanzwe, primer yibanze ya 0.2 μM izatanga ibisubizo byiza;niba imikorere ya reaction ari mibi, kwibanda kwa primer birashobora guhinduka murwego rwa 0.2-1 μM.Ubushishozi bwa probe mubusanzwe butezimbere murwego rwa 0.1-0.3 μM.Ubushakashatsi bwibanze bwibanze burashobora gukorwa kugirango ubone ibyiza bya primer na probe.

Umukino wo gusiganwa ku magare

Inyandiko

1.Igipimo cya amplification ya ADN polymerase yihuta ntigomba kuba munsi ya 1 kb / 10 s.Ubushyuhe bwiyongera nigabanuka, uburyo bwo kugenzura ubushyuhe nuburyo bwo gutwara ubushyuhe bwibikoresho bitandukanye bya PCR biratandukanye cyane, birasabwa rero guhindura uburyo bwiza bwo kwitwara kubikoresho byihariye bya PCR.

2.Sisitemu irahinduka cyane, hamwe nibisobanuro byihariye kandi byunvikana.

3.Birakwiye gukoreshwa nkibyiyumvo bihanitse bya PCR byerekana reagent, kandi birashobora gukoreshwa muri reaction ya multiplex PCR.

4.5 ′ → 3 ′ ibikorwa bya polymerase, 5 ′ → 3 ′ ibikorwa bya exonuc Please;oya 3 ′ → 5 ′ ibikorwa bya exonuclease;nta gikorwa cyo gusuzuma.

5.Birakwiriye kwipimisha ubuziranenge numubare wa PCR na RT-PCR.

6.3 ′ iherezo ryibicuruzwa bya PCR ni A, irashobora gukoronizwa muburyo bwa T.

7.Uburyo butatu bwintambwe burasabwa kuri primers ifite ubushyuhe buke bwa annealing cyangwa kugirango hongerwe ibice birenga 200 bp.