Rnase Inhibitor
Murine RNase inhibitor ndi recombinant murine RNase inhibitor yowonetsedwa ndikuyeretsedwa kuchokera ku E.coli.Imamangiriza ku RNase A, B kapena C mu chiŵerengero cha 1: 1 kupyolera mu mgwirizano wosagwirizanitsa, motero imalepheretsa ntchito ya ma enzyme atatu ndikuteteza RNA kuti isawonongeke.Komabe, sizothandiza motsutsana ndi RNase 1, RNase T1, S1 Nuclease, RNase H kapena RNase kuchokera ku Aspergillus.Murine RNase inhibitor inayesedwa ndi RT-PCR, RT-qPCR ndi IVT mRNA, ndipo inali yogwirizana ndi malonda osiyanasiyana a Reverse transcriptases, DNA polymerases ndi RNA polymerases.
Poyerekeza ndi anthu a RNase inhibitors, murine RNase inhibitor ilibe ma cysteine awiri omwe amakhudzidwa kwambiri ndi okosijeni omwe amachititsa kuti inhibitor iwonongeke.Izi zikupangitsa kuti ikhale yokhazikika pamagawo otsika a DTT (osakwana 1 mM).Izi zimapangitsa kuti zikhale zoyenera kugwiritsidwa ntchito potengera kuchuluka kwa DTT komwe kumatsutsana ndi zomwe zimachitika (mwachitsanzo Real-time RT-PCR).
Akupempha
Izi zitha kugwiritsidwa ntchito kwambiri pakuyesa kulikonse komwe kusokoneza kwa RNase kotheka kupewa kuwonongeka kwa RNA, monga:
1.Kuphatikizika kwa chingwe choyamba cha cDNA, RT-PCR, RT-qPCR, ndi zina zambiri.
2.Imateteza RNA kuti isawonongeke panthawi yolembera/kumasulira (mwachitsanzo, mu vitro viral replication system).
3.Kuletsa ntchito ya RNase panthawi yopatukana ndi kuyeretsedwa kwa RNA.
Zosungirako
Mankhwalawa amatha kusungidwa pa -25～- 15 ℃, zomveka kwa zaka 2.
Bafa yosungirako
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT ndi 50% glycerol.
Tanthauzo la unit
Kuchuluka kwa murine RNase inhibitor yomwe imayenera kulepheretsa ntchito ya 5ng ya ribonuclease A ndi 50% idatanthauzidwa ngati unit imodzi (U).
Molecular kulemera kwa mapuloteni
Kulemera kwa molekyulu ya murine RNase inhibitor ndi 50 kDa.
Kuwongolera khalidwe
Exonuclease Zochita:
40 U ya murine RNase inhibitor yokhala ndi 1 μg λ -Hind III digest DNA pa 37 ℃ kwa maola a 16 sikubala kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Endonuclease ntchito:
40 U ya murine RNase inhibitor yokhala ndi 1μ g λ DNA pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Kuyimba Zochita:
40U ya murine RNase inhibitor yokhala ndi 1μ g pBR322 pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
RNase Zochita:
40U ya murine RNase inhibitor yokhala ndi 1.6μ g MS2 RNA kwa 4h pa 37 ℃ sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
E.coli DNA:
40 U ya murine RNase inhibitor imawunikiridwa kukhalapo kwa E. coli genomic DNA pogwiritsa ntchito TaqMan qPCR yokhala ndi zoyambira za E. coli 16S rRNA locus.E. coli genomic DNA kuipitsidwa ndi ≤ 0.1 pg/40 U.
Notis
1.Kugwedezeka kwachiwawa kapena kugwedezeka kudzachititsa kuti enzyme isagwire ntchito.
2.Kutentha kwabwino kwambiri kwa izi inhibitor inali 25-55 ℃, ndipo Idatsekedwa pa 65 ℃ ndi kupitilira apo.
3.Zochita za RNase H, RNase 1 ndi RNase T1 sizinalepheretsedwe ndi murine RNase inhibitor.
4.Kuletsa ntchito ya RNase kunapezeka mu pH yambiri (pH 5-9 zonse zinali zogwira ntchito), ndipo ntchito yapamwamba kwambiri inawonedwa pa pH 7-8.
5.Popeza ma ribonucleases nthawi zambiri amasunga zochitika m'malo osinthika, kusamala kuyenera kuchitidwa kuti musawononge mamolekyu a RNase Inhibitor omwe amakhala ndi ribonuclease.Pofuna kupewa kutulutsa kwa ribonuclease yogwira, kutentha kopitilira 50 ° C ndi kuchuluka kwa urea kapena ma denaturing agents ayenera kupewa.
