Ihe mgbochi Rnase
Murine RNase inhibitor bụ ihe mgbochi murine RNase egosipụtara ma sachaa ya na E.coli.Ọ na-ejikọta na RNase A, B ma ọ bụ C na nha 1: 1 site na njikọta na-abụghị nke covalent, si otú a na-egbochi ọrụ nke enzymes atọ na ichebe RNA na mmebi.Agbanyeghị, ọ naghị adị irè megide RNase 1, RNase T1, S1 Nuclease, RNase H ma ọ bụ RNase sitere na Aspergillus.A nwalere inhibitor Murine RNase site na RT-PCR, RT-qPCR na IVT mRNA, yana dakọtara na azụmaahịa dị iche iche Reverse transcriptases, DNA polymerases na RNA polymerases.
Tụnyere ndị na-egbochi RNase mmadụ, murine RNase inhibitor enweghị cysteine abụọ nwere mmetụta dị ukwuu na oxidation nke na-akpata inactivation nke inhibitor.Nke ahụ na-eme ka ọ kwụsie ike na obere mkpokọta DTT (ihe na-erughị 1 mM).Njirimara a na-eme ka ọ dabara adaba maka mmeghachi omume ebe oke DTT dị njọ na mmeghachi omume (dịka RT-PCR oge).
Angwa
Enwere ike iji ngwaahịa a mee ihe na nnwale ọ bụla ebe nnyonye anya RNase ga-ekwe omume iji zere mmebi RNA, dị ka:
1.Njikọ nke eriri mbụ nke cDNA, RT-PCR, RT-qPCR, wdg.
2.Na-echebe RNA ka ọ ghara imebi n'oge ntụgharị / ntụgharị in vitro (dịka ọmụmaatụ, sistemụ mmegharị nje in vitro).
3.Mgbochi nke ọrụ RNAse n'oge nkewa RNA na ịdị ọcha.
Ọnọdụ Nchekwa
Enwere ike ịchekwa ngwaahịa ahụ na -25 ~ 15 ℃, dị irè maka afọ 2.
Nchekwa nchekwa
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT na 50% glycerol.
Nkọwa nkeji
Ọnụ ọgụgụ nke murine RNase inhibitor chọrọ iji gbochie ọrụ 5ng nke ribonuclease A site na 50% ka akọwara dị ka otu unit (U).
Ibu arọ nke protein
Arọ molikula nke murine RNase inhibitor bụ 50 kDa.
njikwa mma
Exonuclease Ihe omume:
40 U nke murine RNase inhibitor nwere 1 μg λ -Hind III mgbari DNA na 37 ℃ maka awa 16 anaghị eweta mbibi dị ka agarose gel electrophoresis siri kpebie.
Ọrụ Endonuclease:
40 U nke murine RNase inhibitor nwere 1μ g λ DNA na 37 ℃ maka awa 16 anaghị eweta mbibi ọ bụla dịka agarose gel electrophoresis siri kpebie.
Ịkpọ oku Ihe omume:
40U nke murine RNase inhibitor nwere 1μ g pBR322 na 37 ℃ maka awa 16 anaghị eweta mbibi ọ bụla dịka agarose gel electrophoresis siri kpebie.
RNase Ihe omume:
40U nke murine RNase inhibitor nwere 1.6μ g MS2 RNA maka 4h na 37℃ anaghị amịpụta mmebi dị ka agarose gel electrophoresis kpebiri.
E.coli DNA:
A na-enyocha 40 U nke murine RNase inhibitor maka ọnụnọ nke E. coli genomic DNA na-eji TaqMan qPCR nwere primers kpọmkwem maka ebe E. coli 16S rRNA.Mmetọ DNA nke E.coli bụ ≤ 0.1 pg/40 U.
Notes
1.Oscillation na-eme ihe ike ma ọ bụ mkpali ga-eduga n'ịrụ ọrụ enzyme.
2.The kacha okpomọkụ nso nke a inhibitor bụ 25-55 ℃ , na Ọ e inactivated na 65 ℃ na n'elu.
3. A naghị egbochi ọrụ nke RNase H, RNase 1 na RNase T1 site na murine RNase inhibitor.
4. Achọpụtara ihe mgbochi nke ọrụ RNase n'ọtụtụ pH (pH 5-9 niile na-arụ ọrụ), a na-ahụkwa ọrụ kachasị elu na pH 7-8.
5.Ebe ọ bụ na ribonucleases na-ejigide ọrụ n'okpuru ọnọdụ denaturing, a ghaghị ịkpachara anya iji zere denaturing RNase Inhibitor molecules nke ejirila ribonuclease gbakọta.Iji gbochie ntọhapụ nke ribonuclease na-arụ ọrụ, a ga-ezere okpomọkụ karịrị 50 Celsius C yana nnukwu urea ma ọ bụ ihe ndị ọzọ na-egbochi denatu.
