Rnase Inhibitor
Murine RNase inhibitor ke recombinant murine RNase inhibitor e hlahisitsoeng le ho hloekisoa ho tsoa ho E.coli.E tlama ho RNase A, B kapa C ka karolelano ea 1: 1 ka ho kopanya ho se nang covalent, kahoo e thibela mosebetsi oa li-enzyme tse tharo le ho sireletsa RNA ho senyeha.Leha ho le joalo, ha e sebetse khahlanong le RNase 1, RNase T1, S1 Nuclease, RNase H kapa RNase ho tloha Aspergillus.Murine RNase inhibitor e ile ea lekoa ke RT-PCR, RT-qPCR le IVT mRNA, 'me e ne e lumellana le mefuta e fapaneng ea khoebo ea Reverse transcriptases, DNA polymerases le RNA polymerase.
Ha ho bapisoa le li-inhibitors tsa RNase tsa batho, murine RNase inhibitor ha e na li-cysteine tse nang le maikutlo a matla haholo ho oxidation e bakang ho se sebetse ha inhibitor.Seo se etsa hore e tsitsitse maemong a tlase a DTT (tlase ho 1 mM).Tšobotsi ena e etsa hore e tšoanelehe ho sebelisoa karabelong moo likhakanyo tse phahameng tsa DTT li leng khahlanong le karabelo (mohlala, nako ea 'nete RT-PCR).
Akopo
Sehlahisoa sena se ka sebelisoa haholo tekong efe kapa efe moo tšitiso ea RNase e ka khonehang ho qoba ho senyeha ha RNA, joalo ka:
1.Synthesis ea strand ea pele ea cDNA, RT-PCR, RT-qPCR, joalo-joalo.
2.E sireletsa RNA ho senyeha ha in vitro transcript/phetolelo (mohlala, in vitro viral replication system).
3.Thibelo ea ts'ebetso ea RNase nakong ea karohano le tlhoekiso ea RNA.
Maemo a polokelo
Sehlahisoa se ka bolokoa ho -25～- 15 ℃, se sebetsa lilemo tse peli.
Sebaka sa polokelo
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT le 50% glycerol.
Tlhaloso ea yuniti
Palo ea murine RNase inhibitor e hlokahalang ho thibela mosebetsi oa 5ng oa ribonuclease A ka 50% e ne e hlalosoa e le karolo e le 'ngoe (U).
Boima ba limolek'hule tsa protheine
Boima ba molek'hule ea murine RNase inhibitor ke 50 kDa.
Taolo ea boleng
Exonuclease Ketsahalo:
40 U ea murine RNase inhibitor e nang le 1 μg λ -Hind III digest DNA ho 37 ℃ bakeng sa lihora tsa 16 ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
Ts'ebetso ea Endonuclease:
40 U ea murine RNase inhibitor e nang le 1μ g λ DNA ho 37 ℃ bakeng sa lihora tsa 16 ha e fane ka ho senyeha ha ho khethoa ke agarose gel electrophoresis.
Ho thala Ketsahalo:
40U ea murine RNase inhibitor e nang le 1μ g pBR322 ho 37 ℃ bakeng sa lihora tsa 16 ha e fane ka ho senyeha ha ho khethoa ke agarose gel electrophoresis.
RNase Ketsahalo:
40U ea murine RNase inhibitor e nang le 1.6μ g MS2 RNA bakeng sa 4h ho 37 ℃ ha e fane ka ho senyeha ha ho khethoa ke agarose gel electrophoresis.
E.coli DNA:
40 U ea murine RNase inhibitor e hlahlojoa bakeng sa boteng ba E. coli genomic DNA e sebelisa TaqMan qPCR e nang le li-primers tse khethehileng bakeng sa sebaka sa E. coli 16S rRNA.Tšilafalo ea E. coli genomic DNA ke ≤ 0.1 pg/40 U.
Notes
1.Oscillation e mabifi kapa ho sisinyeha ho tla lebisa ho inctivation ea enzyme.
2. The optimum mocheso mefuta e fapaneng ya inhibitor ena e ne e le 25-55 ℃ , 'me E ne e inactivated ho 65 ℃ le holimo.
3. Mesebetsi ea RNase H, RNase 1 le RNase T1 ha ea ka ea thibeloa ke murine RNase inhibitor.
4. Thibelo ea ts'ebetso ea RNase e fumanoe ka mefuta e mengata ea pH (pH 5-9 kaofela e ne e sebetsa), 'me mosebetsi o phahameng ka ho fetisisa o ile oa hlokomeloa ho pH 7-8.
5. Kaha li-ribonuclease hangata li boloka ts'ebetso tlas'a maemo a denaturing, tlhokomelo e tlameha ho nkoa ho qoba ho thibela limolek'hule tsa RNase Inhibitor tse kopantsoeng le ribonuclease.Ho thibela ho lokolloa ha ribonuclease e sebetsang, mocheso o moholo ho feta 50 ° C le likhahla tse phahameng tsa urea kapa lisebelisoa tse ling tsa denaturing li lokela ho qojoa.
