Robustart Taq DNA Polymerase

Robustart Taq DNA Polymerase ndi chiyambi chotentha cha DNA polymerase.Izi sizingangoletsera bwino zomwe zimachitika chifukwa cha kusakhazikika kwapadera kwa zoyambira kapena kuphatikizira koyambira pokonzekera ndi kukulitsa dongosolo la PCR.Chifukwa chake, ili ndi tsatanetsatane wabwino kwambiri ndipo imakhala yothandiza kwambiri pakukulitsa ma tempuleti otsika, ndipo ndiyoyenera kuchulukitsa ma PCR amplification.Kuphatikiza apo, mankhwalawa ali ndi magwiridwe antchito abwino kwambiri, ndipo zotsatira zokhazikika zokulitsa zitha kupezeka mumitundu yosiyanasiyana yamachitidwe a PCR.

Zigawo

1.5 U/μL Robustart Taq DNA polymerase

2.10 × PCR Buffer II (Mg²+ yaulere) (ngati mukufuna)

3.25 mm MgCl₂(posankha)

* 10 × PCR Buffer II (Mg²+ yaulere) ilibe dNTP ndi Mg²+, chonde onjezani dNTPs ndi MgCl₂pokonzekera kachitidwe kachitidwe.

Mapulogalamu Ovomerezeka

1.Kukulitsa mwachangu.

2.Kuchulukitsa kangapo.

3.Kuchulukitsidwa kwachindunji kwa magazi, swabs, ndi zitsanzo zina.

4.Kuzindikira matenda opuma.

Mkhalidwe Wosungira

-20 ° C posungira nthawi yayitali, ziyenera kusakanizidwa bwino musanagwiritse ntchito, pewani kuzizira pafupipafupi.

*Ngati mvula ikagwa pambuyo pa firiji, ndi bwino;tikulimbikitsidwa kuti tigwirizane ndi kutentha kwa chipinda musanayambe kusakaniza ndi kugwiritsa ntchito.

Tanthauzo la Chigawo

Chigawo chimodzi chogwira ntchito (U) chimatanthauzidwa ngati kuchuluka kwa enzyme yomwe imaphatikizapo 10 nmol ya deoxyribonucleotide mu zinthu zosasungunuka za asidi pa 74 ° C kwa 30mins pogwiritsa ntchito activated salmon umuna DNA monga template/primer.

Kuwongolera Kwabwino

1.SDS-PAGE electrophoretic chiyero choposa 98%.

2.Kukhudzika kukhudzika, kuwongolera kwa batch-to-batch, kukhazikika.

3.Palibe exogenous nuclease ntchito, palibe exogenous endonuclease kapena exonuclease kuipitsidwa

Malangizo

Kukonzekera kwa Reaction

Ndemanga:

1) a.Buffer ilibe dNTP ndi Mg²+, chonde onjezani dNTPs ndi MgCl₂pokonzekera kachitidwe kachitidwe.

2) b.Ngati agwiritsidwa ntchito pa qPCR/qRT-PCR, ma probe a fulorosenti ayenera kuwonjezeredwa pamachitidwe.Kawirikawiri, chigawo chomaliza cha 0.2 μM chidzapereka zotsatira zabwino;Ngati zomwe zimachitika ndizovuta, ndende yoyambira imatha kusinthidwa mumitundu ya 0.2-1 μM.The kafukufuku ndende zambiri wokometsedwa mu osiyanasiyana 0.1-0.3 μM.Kuyesa kwa concentration gradient kumatha kuchitidwa kuti mupeze kuphatikiza kwabwino kwambiri kwa primer ndi probe.

Thermal cycling protocol

Zolemba

1.Kuchulukitsa kwa DNA polymerase mwachangu sikuyenera kuchepera 1 kb/10 s.Kutentha kwa kukwera ndi kugwa kwa kutentha, mawonekedwe a kutentha ndi kutentha kwa zipangizo zosiyanasiyana za PCR zimasiyana kwambiri, choncho tikulimbikitsidwa kuti tikwaniritse momwe zinthu zilili pa chipangizo cha PCR.

2.Dongosololi ndi losinthika kwambiri, lokhala ndi chidziwitso chambiri komanso chidwi.

3.Zoyenera kugwiritsidwa ntchito ngati ma reagents ozindikira kwambiri a PCR, ndipo atha kugwiritsidwa ntchito muzochita zokulitsa za PCR.

4.5′→3′ polymerase ntchito, 5′→3′ exonuclease ntchito;palibe 3′→5′ ntchito exonuclease;palibe ntchito yowerengera.

5.Oyenera kuyezetsa koyenera komanso kuchuluka kwa PCR ndi RT-PCR.

6.Mapeto a 3' a PCR ndi A, omwe amatha kupangidwa mwachindunji kukhala T vector.

7.Njira ya masitepe atatu imalimbikitsidwa pazida zoyambira zomwe zimakhala ndi kutentha pang'ono kapena kukulitsa zidutswa zotalika kuposa 200 bp.