mRNA Cap2'-O-Methyltransferase

mRNA Cap 2´ -O-methyltransferase yakomotse kumurongo wa recombinant E. coli itwara gene yinkingo mRNA Cap 2 ´-O-Methyltransferase.Iyi misemburo yongeramo itsinda rya methyl kumwanya wa 2´-O ya nucleotide ya mbere ijyanye nimiterere ya cap kuri 5´end ya RNA. Enzyme ikoresha S-adenosylmethionine (SAM) nkumuterankunga wa methyl kuri methylate yafashwe RNA (cap -0) bivamo imiterere ya cap- 1.

Imiterere ya Cap1 irashobora kongera imikorere yubusobanuro, igatezimbere imvugo ya mRNA muguhindura no kugerageza microinjection.Iyi misemburo isaba byumwihariko RNA ifite capa ya m7GpppN nka substrate.Ntishobora gukoresha RNA hamwe na pN, ppN, pppN cyangwa GpppN kumpera ya 5´.RNA ifashwe irashobora gutegurwa hifashishijwe vitro transcription ukoresheje cap analog cyangwa ukoresheje capping enzymatique ukoresheje Enzyme ya Vaccinia.

Ibigize

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

10 × Gufata reaction ya Buffer

Ububiko

-25 ～ - 15 ℃ kubika （Irinde gusubiramo inshuro nyinshi gukonjesha haw

Ububiko

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.

Igisobanuro cyibice

Igice kimwe gisobanurwa nkubunini bwa enzyme isabwa kuri methylate pmoles 10 za 80 nt zafashwe na RNA mumasaha 1 kuri 37 ° C.

Kugenzura ubuziranenge

Exonuc Please: 50U ya mRNA Cap 2 ´ -O-Methyltransferase hamwe na 1μg λ-Hind III igogora ADN kuri 37 ℃ kumasaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.

Endonuc Please: 50 U ya mRNA Cap 2 ´ -O-Methyltransferase hamwe na 1 μg λDNA kuri 37 ℃ kumasaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.

Nickase: 50U ya mRNA Cap 2 ´ -O-Methyltransferase hamwe na 1μg pBR322 kuri 37 ℃ kumasaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.

RNase: 50U ya mRNA Cap 2 ´ -O-Methyltransferase hamwe na 1,6μg MS2 RNA mumasaha 4 kuri 37 ℃ itanga umusaruro mubi nkuko byagenwe na agarose gel electrophorei.

E. coli ADN: 50U ya mRNA Cap 2 ´ -O-Methyltransferase irerekanwa kugirango ihariE. coli ADN genomic ukoresheje TaqMan qPCR hamwe na primers yihariye yaE. coli 16S rRNA.UwitekaE. coli genomic ADN yanduye ni = 1E. coli genome.

Indwara ya bagiteri Endotoxin: Ikizamini cya LAL, ukurikije Ubushinwa Pharmacopoeia IV 2020, uburyo bwo gupima gel, uburyo rusange (1143).Indwara ya bagiteri igomba kuba = 10 EU / mg.

Sisitemu yo kwitwara hamwe nibisabwa

1. Huza umubare ukwiye wa Capped RNA na RNase idafite RN2 muri tike ya microfuge 1.5 mL kugeza mubunini bwa 16.0 µL.

2. Shyushya kuri 65 ℃ muminota 5 ukurikirwa no kwiyuhagira urubura muminota 5.

3. Ongeramo ibice bikurikira muburyo bwateganijwe (kuri methylation ya Capped RNA

munsi ya 10

* 10.

4. Incubate kuri 37 ℃ kumasaha 1 (amasaha 2 ya incubation irasabwa kubice bigenewe munsi ya 200 nt).

Porogaramu

Kunoza imvugo ya mRNA mugihe cya microinjection no kugerageza.

Inyandiko zikoreshwa

1. Mbere yo kubyitwaramo, RNA igomba kwezwa no gushonga mumazi adafite nuclease, ibisubizo byose ntibigomba kuba birimo EDTA na ion.

2. Birasabwa gushyushya icyitegererezo RNA kuri 65 ℃ kuminota 5 mbere yuko reaction yo gukuraho imiterere ya kabiri kuri 5´end ya transcript.Irashobora kwagurwa kugeza kuminota 10 kumurongo 5 ´ -imiterere yimiterere.