Robustart Taq DNA Polymerase

Robustart Taq DNA Polymerase ke qalo e chesang ea DNA polymerase.Sehlahisoa sena ha se khone ho thibela feela karabelo e sa tobang e bakiloeng ke ho kopanngoa ho sa tobang ha li-primers kapa ho kopanya li-primer nakong ea tokiso ea sistimi ea PCR le ho holisa.Ka hona, e na le boits'oaro bo botle haholo 'me e sebetsa hantle bakeng sa ho holisa litempele tse tlase tsa mahloriso,' me e loketse karabelo ea amplification ea PCR e mengata.Ho feta moo, sehlahisoa sena se sebetsa hantle haholo, 'me liphetho tse tsitsitseng tsa amplification li ka fumanoa ka mefuta e fapaneng ea karabelo ea PCR.

Likaroloana

1.5 U/μL Robustart Taq DNA polymerase

2.10 × PCR Buffer II (Mg²+ mahala) (ho ikhethela)

3.25 limilimithara MgCl₂(ka boikhethelo)

* 10 × PCR Buffer II (Mg²+ mahala) ha e na dNTP le Mg²+, ka kopo eketsa dNTPs le MgCl₂ha u lokisetsa mokhoa oa ho arabela.

Likopo tse khothalelitsoeng

1.Ho phahamisa kapele.

2.Amplification tse ngata.

3.Ho holisa mali ka kotloloho, li-swabs le lisampole tse ling.

4.Ho lemoha mafu a ho hema.

Boemo ba polokelo

-20°C bakeng sa polokelo ea nako e telele, e lokela ho tsoakoa hantle pele e sebelisoa, qoba ho qhibiliha khafetsa.

*Haeba pula e na ka mor'a sehatsetsi, ho tloaelehile;ho kgothaletswa ho leka-lekanya mocheso oa kamore pele o kopanya le ho sebelisa.

Tlhaloso ea Yuniti

Karolo e le 'ngoe e sebetsang (U) e hlalosoa e le palo ea enzyme e kenyelletsang 10 nmol ea deoxyribonucleotide ka har'a lintho tse sa keneng acid ka 74°C bakeng sa 30mins ho sebelisoa DNA ea peō ea salmon e entsoeng e le template/primer.

Taolo ea Boleng

1.SDS-PAGE electrophoretic purity e kholo ho feta 98%.

2.Amplification sensitivity, taolo ea batch-to-batch, botsitso.

3.Ha ho ts'ebetso ea "nuclease exogenous", ha ho na endonuclease ea kantle kapa tšilafalo ea exonuclease.

Litaelo

Rection Setup

Lintlha:

1) a.Buffer ha e na dNTP le Mg²+, ka kopo eketsa dNTPs le MgCl₂ha u lokisetsa mokhoa oa ho arabela.

2) b.Haeba e sebelisetsoa qPCR/qRT-PCR, lisebelisoa tsa fluorescent li lokela ho eketsoa tsamaisong ea karabelo.Hangata, palo ea ho qetela ea 0,2 μM e tla fana ka liphello tse ntle;haeba ts'ebetso ea karabelo e fokola, mohopolo oa primer o ka fetoloa maemong a 0.2-1 μM.Hangata mahloriso a probe a ntlafatsoa maemong a 0.1-0.3 μM.Liteko tsa Concentration gradient li ka etsoa ho fumana motsoako o motle ka ho fetisisa oa primer le probe.

Thermal cycling protocol

Lintlha

1.Sekhahla sa amplification ea DNA polymerase e potlakileng ha ea lokela ho ba ka tlase ho 1 kb/10 s.Sekhahla sa ho nyoloha le ho theoha ha mocheso, mokhoa oa ho laola mocheso le katleho ea mocheso oa lisebelisoa tse fapaneng tsa PCR li fapana haholo, kahoo ho kgothaletswa ho ntlafatsa maemo a nepahetseng a karabelo bakeng sa sesebelisoa se khethehileng sa PCR.

2.Sistimi e khona ho ikamahanya le maemo haholo, e na le boits'oaro bo phahameng le kutloisiso.

3.E loketse ho sebelisoa e le li-reagents tsa ho lemoha tse phahameng tsa PCR, 'me e ka sebelisoa ho karabelo ea amplification ea PCR ea multiplex.

4.5′→3′ mosebetsi oa polymerase, 5′→3′ mosebetsi oa exonuclease;ha ho 3′→5′ mosebetsi oa exonuclease;ha ho mosebetsi oa ho bala.

5.E loketse tlhahlobo ea boleng le palo ea PCR le RT-PCR.

6.Qetello ea 3' ea sehlahisoa sa PCR ke A, e ka etsoang ka ho toba ho T vector.

7.Mokhoa oa mehato e meraro o khothaletsoa bakeng sa li-primers tse nang le mocheso o tlase oa annealing kapa bakeng sa ho holisa likhechana tse telele ho feta 200 bp.