I-Rnase Inhibitor (iGlycerol yasimahla)
I-Murine RNase inhibitor yi-recombinant murine RNase inhibitor echazwe kwaye ihlanjululwe kwi-E.coli.Ibophelela kwi-RNase A, B okanye C kwi-1: umlinganiselo we-1 ngokusebenzisa i-non-covalent bonding, ngaloo ndlela inqanda umsebenzi wee-enzyme ezintathu kunye nokukhusela i-RNA ekuthotyweni.Nangona kunjalo, ayisebenzi ngokuchasene ne-RNase 1, i-RNase T1, i-S1 Nuclease, i-RNase H okanye i-RNase esuka kwi-Aspergillus.I-Murine RNase inhibitor yavavanywa yi-RT-PCR, RT-qPCR kunye ne-IVT mRNA, kwaye yayihambelana neentlobo ezahlukeneyo zentengiso ye-Reverse transcriptases, i-DNA polymerases kunye ne-RNA polymerases.
Xa kuthelekiswa ne-inhibitors ye-RNase yabantu, i-murine RNase inhibitor ayinayo i-cysteines ezimbini ezithintekayo kakhulu kwi-oxidation ebangela ukungasebenzi kwe-inhibitor.Oko kwenza ukuba izinze kwiindawo eziphantsi ze-DTT (ngaphantsi kwe-1 mM).Olu phawu luyenza ilungele ukusetyenziswa kwiimpendulo apho ugxininiso oluphezulu lwe-DTT luchasene nokusabela (umz. Ixesha lokwenyani i-RT-PCR).
Aisicelo
Le mveliso inokusetyenziswa ngokubanzi kulo naluphi na uvavanyo apho uphazamiseko lwe-RNase lunokwenzeka ukunqanda ukuthotywa kwe-RNA, efana nale:
I-1.First-strand cDNA synthesis, RT-PCR, RT-qPCR, njl.;
2.Khusela i-RNA ekuthotyweni kwi-in-vitro transcription/uguqulelo (umz. ukuphindaphindwa kwentsholongwane kwi-vitro);
3.Ukuvinjelwa komsebenzi we-RNase ngexesha lokuzihlukanisa kwe-RNA kunye nokuhlanjululwa.
Iimeko zokuGcina
Gcina kwi -25～-15℃;
Imijikelo ye-Freeze-thaw ≤ amaxesha ama-5;
Isebenza iminyaka eyi-1.
Inkcazo yeyunithi
Iyunithi enye ichazwa njengexabiso le-RNase Inhibitor efunekayo ukuthintela umsebenzi we-5 ng ye-RNAse A ngama-50%.
Ubunzima bemolekyuli
I-RNase Inhibitor (i-Glycerol-free) yiprotheni ye-50 kDa.
Ulawulo lwemeko
I-Exonuclease Umsebenzi:
Ukufakwa kwe-40 U ye-enzyme kunye ne-1 μg λ-Hind III yokugaya i-DNA kwiiyure ze-16 kwi-37 ° C kubangele kungabikho ukuthotywa okubonakalayo kwe-DNA njengoko kunqunywe yi-gel electrophoresis.
Umsebenzi we-Endonuclease:
Ukufakwa kwe-40 U ye-enzyme kunye ne-1 μg λ DNA kwiiyure ze-16 kwi-37 ° C kubangele kungabikho ukuthotywa okubonakalayo kwe-DNA njengoko kunqunywe yi-gel electrophoresis.
Ukuqhula Umsebenzi:
Ukufakwa kwe-40 U ye-enzyme kunye ne-1 μg pBR322 kwiiyure ze-16 kuma-37℃ kubangele kungabikho kuthotywa kwe-DNA njengoko kumiselwe yi-gel electrophoresis.
RNase Umsebenzi:
Ukufakwa kwe-40 U ye-enzyme kunye ne-1.6 μg MS2 RNA kwiiyure ezi-4 kuma-37℃ kubangele kungabikho ukuthotywa okubonakalayo kwe-RNA njengoko kumiselwe yi-gel electrophoresis.
E.coli DNA:
I-40 U ye-Enzyme ichongiwe yi-TaqMan qPCR.I-E.coli DNA yi ≤ 0. 1pg/40U.
Notes
1.Musa ukungcangcazela okanye uvuthulule ngamandla ukuthintela ukungasebenzi kwe-enzyme.
I-2.RNase inhibitor iyasebenza kumaqondo okushisa ukusuka kwi-25 ℃ ukuya kwi-55 ℃ kwaye ayisebenzi kwi-≥65℃.
3.Umsebenzi we-RNase H, i-RNase 1, kunye ne-RNase T1 awuthintelwa.
I-4.Uluhlu lwe-pH lokuthintela umsebenzi we-RNase lubanzi (lusebenza kwi-pH 5-9), lubonisa umsebenzi ophezulu kwi-pH 7-8.
