mRNA Cap2'-O-Methyltransferase

mRNA Cap 2'-O-methyltransferase an samo shi daga recombinant E. coli iri wanda ke dauke da kwayar halitta don maganin rigakafi mRNA Cap 2 '-O-Methyltransferase.Wannan enzyme yana ƙara ƙungiyar methyl a matsayi na 2'-O na farkon nucleotide kusa da tsarin cap a 5'karshen RNA. Enzyme yana amfani da S-adenosylmethionine (SAM) a matsayin mai ba da gudummawar methyl zuwa methylate capped RNA ( hula -0) yana haifar da cap- 1 tsarin.

Tsarin Cap1 na iya ƙara ingantaccen fassarar fassarar, inganta bayyanar mRNA a cikin canzawa da gwaje-gwajen microinjection. Wannan enzyme na musamman yana buƙatar RNA tare da m7GpppN cap a matsayin substrate.Ba zai iya amfani da RNA tare da pN, ppN, pppN ko GpppN a ƙarshen 5′.Za a iya shirya RNA da aka rufe ta hanyar rubutun in vitro ta amfani da analog cap ko ta hanyar enzymatic capping ta amfani da Vaccinia Capping Enzyme.

Abubuwan da aka gyara

mRNA Cap 2'-O-Methyltransferase (50U/μL)

10× Capping Reaction Buffer

Adana

-25 ～- 15 ℃ don ajiya (A guji maimaita daskare-narke hawan keke)

Ma'ajiyar ajiya

20 mM Tris-HCl (pH 8.0, 25 ℃), 100 mM NaCl, 1 mM DTT, 0. 1 mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Ma'anar raka'a

An bayyana raka'a ɗaya azaman adadin enzyme da ake buƙata don methylate 10 pmoles na 80 nt capped RNA kwafin a cikin awa 1 a 37°C.

Gwajin kula da inganci

Exonuclease: 50U na mRNA Cap 2 '-O-Methyltransferase tare da 1μg λ-Hind III narke DNA a 37 ℃ na 16 hours ba ya haifar da lalacewa kamar yadda aka ƙaddara ta hanyar agarose gel electrophoresis.

Endonuclease: 50 U na mRNA Cap 2 '-O-Methyltransferase tare da 1 μg λDNA a 37 ℃ na 16 hours ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

Nickase: 50U na mRNA Cap 2 '-O-Methyltransferase tare da 1μg pBR322 a 37 ℃ na sa'o'i 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

RNase: 50U na mRNA Cap 2 '-O-Methyltransferase tare da 1.6μg MS2 RNA na 4 hours a 37 ℃ yana haifar da rashin lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

E. coli DNA: 50U na mRNA Cap 2 '-O-Methyltransferase an duba shi don kasancewarE. coli DNA na genomic ta amfani da TaqMan qPCR tare da abubuwan da aka keɓance na musamman donE. coli 16S rRNA wuri.TheE. coli Kwayoyin halittar DNA shine = 1E. coli kwayoyin halitta.

Kwayoyin cuta Endotoxin: LAL-gwajin, bisa ga Sin Pharmacopoeia IV 2020 edition, gel iyaka gwajin hanya, general doka (1143).Ya kamata abun ciki na endotoxin na ƙwayoyin cuta ya zama = 10 EU/mg.

Tsarin amsawa da yanayi

1. Haɗa adadin da ya dace na Capped RNA da H2O mara RNase a cikin bututun microfuge 1.5 ml zuwa ƙarar ƙarshe na 16.0 µL.

2. Gasa a 65 ℃ na minti 5 sannan a yi wanka na kankara na minti 5.

3. Ƙara abubuwan da ke biyo baya a cikin tsari da aka ƙayyade (don methylation na Capped RNA

kasa da 10

* 10 × Capping Reaction Buffer: 500 mM Tris-HCl (pH 8.0, 25 ℃), 50 mM KCl, 10 mM MgCl2, 10 mM DTT.

4. Incubate a 37 ℃ na 1 hour (2 hours shiryawa ne shawarar ga manufa guntu kasa da 200 nt).

Aikace-aikace

Don inganta maganganun mRNA yayin gwajin microinjection da canzawa.

Bayanan kula akan amfani

1. Kafin amsawa, RNA ya kamata a tsarkake kuma a narkar da shi a cikin ruwa maras kyau, duk mafita bai kamata ya ƙunshi EDTA da ions ba.

2. Ana bada shawara don zafi samfurin RNA a 65 ℃ na 5 min kafin amsawa don cire tsarin na biyu akan 5'karshen rubutun.Ana iya tsawaita shi zuwa mintuna 10 don hadadden tsari na 5 ′-terminal.