Mai hana Rnase
Murine RNase inhibitor shine mai hana murine RNase wanda aka bayyana kuma an tsarkake shi daga E.coli.Yana ɗaure zuwa RNase A, B ko C a cikin rabo na 1:1 ta hanyar haɗin kai mara daidaituwa, ta haka yana hana ayyukan enzymes uku da kare RNA daga lalacewa.Koyaya, Ba shi da tasiri akan RNase 1, RNase T1, S1 Nuclease, RNase H ko RNase daga Aspergillus.Murine RNase inhibitor an gwada ta RT-PCR, RT-qPCR da IVT mRNA, kuma ya dace da nau'ikan bayanan Reverse na kasuwanci, polymerases na DNA da RNA polymerases.
Idan aka kwatanta da masu hana RNase na ɗan adam, mai hana RNase na murine ba ya ƙunshi nau'in cysteines guda biyu waɗanda ke da matukar damuwa ga oxidation wanda ke haifar da rashin kunna mai hanawa.Wannan yana mai da shi kwanciyar hankali a ƙananan adadin DTT (kasa da 1 mM).Wannan fasalin ya sa ya dace don amfani a cikin halayen inda babban taro na DTT ke da illa ga abin da ya faru (misali RT-PCR na ainihi).
Aaikace-aikace
Ana iya amfani da wannan samfur ko'ina a kowane gwaji inda tsangwama na RNase zai yiwu don guje wa lalatawar RNA, kamar:
1.Ƙirar farko na cDNA, RT-PCR, RT-qPCR, da dai sauransu.
2.Yana Kare RNA daga lalacewa yayin rubutawa/fassarar in vitro (misali, in vitro viral replication system).
3.Hana ayyukan RNase yayin rabuwar RNA da tsarkakewa.
Yanayin Ajiya
Ana iya adana samfurin a -25 ℃ - 15 ℃, yana aiki na shekaru 2.
Ma'ajiyar ajiya
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT da 50% glycerol.
Ma'anar raka'a
Adadin murine RNase inhibitor da ake buƙata don hana ayyukan 5ng na ribonuclease A ta 50% an bayyana shi azaman raka'a ɗaya (U).
Nauyin kwayoyin furotin
Nauyin kwayoyin halitta na murine RNase inhibitor shine 50 kDa.
Kula da inganci
Exonuclease Ayyuka:
40 U na murine RNase inhibitor tare da 1 μg λ -Hind III narke DNA a 37 ℃ na sa'o'i 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.
Ayyukan Endonuclease:
40 U na murine RNase inhibitor tare da 1μ g λ DNA a 37 ℃ na 16 hours ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.
Yin waƙa Ayyuka:
40U na murine RNase inhibitor tare da 1μ g pBR322 a 37 ℃ na sa'o'i 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.
RNase Ayyuka:
40U na murine RNase inhibitor tare da 1.6μ g MS2 RNA don 4h a 37 ℃ ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.
E.coli DNA:
40 U na murine RNase inhibitor an duba shi don kasancewar E. coli genomic DNA ta amfani da TaqMan qPCR tare da abubuwan da suka dace don E. coli 16S rRNA locus.Kwayoyin halittar E. coli na DNA shine ≤ 0.1 pg/40 U.
Notes
1.Volent oscillation ko motsawa zai haifar da rashin kunna enzyme.
2.The ganiya zafin jiki kewayon wannan mai hanawa shi ne 25-55 ℃ , kuma An kunna shi a 65 ℃ da sama.
3. Ayyukan RNase H, RNase 1 da RNase T1 ba su hana murine RNase inhibitor.
4.An gano hana aikin RNase a cikin nau'i mai yawa na pH (pH 5-9 duk suna aiki), kuma an lura da mafi girman aiki a pH 7-8.
5.Tun da ribonucleases yawanci yana riƙe da aiki a ƙarƙashin yanayin rashin ƙarfi, dole ne a kula da shi don guje wa lalata ƙwayoyin ƙwayoyin cuta na RNase Inhibitor waɗanda suka haɗu tare da ribonuclease.Don hana fitowar ribonuclease mai aiki, yanayin zafi sama da 50 ° C da yawan adadin urea ko wasu abubuwan cirewa ya kamata a guji.
