Rnase Inhibitor
Murine RNase inhibitor Ko te recombinant murine RNase inhibitor whakapuakihia, ka purea i E.coli.Ka herea ki te RNase A, B, C ranei i roto i te 1:1 ōwehenga na roto i te hononga kore-covalent, na reira ka aukati i te mahi o nga enzymes e toru me te tiaki i te RNA mai i te paheketanga.Engari, Kaore e whai hua ki te RNase 1, RNase T1, S1 Nuclease, RNase H, RNase ranei mai i Aspergillus.I whakamatauria te aukati Murine RNase e te RT-PCR, RT-qPCR me te IVT mRNA, a he hototahi ki nga momo whakawhitinga whakamuri arumoni, DNA polymerases me RNA polymerases.
Ki te whakatauritea ki nga kaitautoko RNase tangata, karekau he rua cysteinees e uru ana te aukati RNase murine e tino aro nui ana ki te waiora e puta ai te koretake o te aukati.Ka noho pumau ki te iti o te DTT (iti iho i te 1 mM).Ko tenei ahuatanga e pai ana mo te whakamahi i nga tauhohenga ka nui te kino o te DTT ki te tauhohenga (hei tauira, RT-PCR-Wa-Tuturu).
Application
Ka taea te whakamahi whanui i tenei hua i roto i nga whakamatautau ka taea te wawaotanga RNase ki te karo i te paheketanga o te RNA, penei:
1.Te whakahiatotanga o te whenu tuatahi o cDNA, RT-PCR, RT-qPCR, etc.
2.Ka parea te RNA mai i te paheketanga i te wa o te whakamaoritanga/whakamaoritanga in vitro (hei tauira, te punaha tukurua viral in vitro).
3.Ko te whakakore i te mahi RNase i te wa o te wehenga RNA me te purenga.
Nga Tikanga Rokiroki
Ka taea te rongoa te hua ki te -25～- 15 ℃, whai mana mo te 2 tau.
Pupuri putunga
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT me te 50% glycerol.
Te whakamaramatanga o te waeine
Ko te nui o te aukati RNase murine e hiahiatia ana hei aukati i te mahi o te 5ng o ribonuclease A na te 50% i tautuhia hei waeine kotahi (U).
Te taumaha ngota o te pūmua
Ko te taumaha ngota o te aukati RNase murine he 50 kDa.
Mana kounga
Exonuclease Mahi:
40 U o murine RNase inhibitor me te 1 μg λ -Hind III digest DNA i te 37 ℃ mo nga haora 16 kaore he whakahekenga i whakatauhia e te electrophoresis gel agarose.
Mahi Endonuclease:
Ko te 40 U o murine RNase inhibitor me te 1μ g λ DNA i te 37 ℃ mo nga haora 16 kaore he whakahekenga ka whakatauhia e te electrophoresis gel agarose.
Te tima Mahi:
Ko te 40U o te aukati RNase murine me te 1μ g pBR322 i te 37 ℃ mo nga haora 16 karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.
RNase Mahi:
Ko te 40U o te aukati RNase murine me te 1.6μ g MS2 RNA mo te 4h i te 37 ℃ kaore he whakahekenga e whakatauhia ana e te electrophoresis gel agarose.
E.coli DNA:
Ko te 40 U o te aukati RNase murine ka tirotirohia mo te aroaro o te E. coli genomic DNA ma te whakamahi i te TaqMan qPCR me nga tohu tuatahi mo te waahi E. coli 16S rRNA.Ko te pokenga DNA genomic E. coli he ≤ 0.1 pg/40 U.
Notes
1.Ko te oscillation tutu, ko te whakakorikori ranei ka arai ki te koretake o te enzyme.
2.Ko te awhe pāmahana tino pai o tenei ko te aukati ko te 25-55 ℃ , a ka whakakorehia i te 65 ℃ me runga ake.
3.Ko nga mahi a RNase H, RNase 1 me RNase T1 kaore i whakakorehia e te aukati RNase murine.
4. Ko te aukati o te mahi RNase i kitea i roto i te whānuitanga o te pH (he kaha katoa te pH 5-9), a ko te mahi teitei i kitea i te pH 7-8.
5.Na te mea ka mau tonu nga ribonucleases i nga mahi i raro i nga tikanga whakaheke, me tupato ki te karo i te whakakore i nga ngota ngota aukati RNase kua uaua ki te ribonuclease.Hei aukati i te tukunga o te ribonuclease hohe, me karohia te pāmahana nui ake i te 50 °C me te kukū teitei o te Urea me etahi atu mea whakaheke.
