mRNA Cap2'-O-Methyltransferase

mRNA Cap 2'-O-methyltransferase waxaa laga soo qaatay dib-u-soo-kabashada E. coli oo sidda hidda-wadaha tallaalka mRNA Cap 2 ′-O-Methyltransferase.Enzyme Tani waxay ku darsataa koox methyl ah booska 2'-O ee nucleotide ugu horreeya ee ku dheggan qaab dhismeedka koofiyadda ee 5'dhamaadka RNA. Ensaymku wuxuu isticmaalaa S-adenosylmethionine (SAM) oo ah deeq-bixiye methyl si uu methylate u daboolo RNA -0) taasoo keentay cap- 1 qaab dhismeed.

Qaab dhismeedka Cap1 wuxuu kordhin karaa hufnaanta tarjumaadda, hagaajinta muujinta mRNA ee gudbinta iyo tijaabooyinka microinjection-ka.Uma isticmaali karo RNA pN, ppN, pppN ama GpppN dhamaadka 5′.RNA la daboolay waxaa lagu diyaarin karaa iyada oo loo marayo qoraalka in vitro iyadoo la isticmaalayo koofiyada analoogga ah ama iyada oo la isticmaalayo koofiyad enzymatic iyadoo la isticmaalayo Vaccinia Capping Enzyme.

Qaybaha

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

10×Capping Reaction Buffer

Kaydinta

-25 ～- 15 ℃ si loo kaydiyo (iska ilaali wareegyada qaboojinta ee soo noqnoqda)

Kaydinta kaydinta

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.

Qeexitaanka unugga

Hal unug ayaa lagu qeexay cadadka insaymiska looga baahan yahay methylate 10 pmoles oo ah 80 nt koob oo qoraal RNA ah gudaha 1 saac 37°C.

Qiimaynta xakamaynta tayada

Exonuclease: 50U of mRNA Cap 2 '-O-Methyltransferase oo leh 1μg λ-Hind III dheefshiidka DNA at 37 ℃ 16 saacadood ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.

Endonuclease: 50 U of mRNA Cap 2 '-O-Methyltransferase oo leh 1 μg λDNA at 37℃ 16 saacadood ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.

Nickase: 50U of mRNA Cap 2 '-O-Methyltransferase oo leh 1μg pBR322 at 37 ℃ 16 saacadood ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.

RNase: 50U of mRNA Cap 2 '-O-Methyltransferase oo leh 1.6μg MS2 RNA 4 saacadood at 37 ℃ ma keeno hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.

E. coli DNA: 50U of mRNA Cap 2 ′-O-Methyltransferase waxaa lagu baaray joogitaankaE. coli DNA-ga genomic iyadoo la adeegsanayo TaqMan qPCR oo leh qalab asal ah oo u gaar ahE. coli 16S rRNA goobta.TheE. coli wasakheynta DNA-da genomic waa =1E. coli genome.

Bakteeriya Endotoxin: LAL-tijaabo, sida uu qabo Chinese Pharmacopoeia IV daabacaadda 2020, gel xadka xadka habka, xeerka guud (1143).Bakteeriyada endotoxin waa inay noqotaa = 10 EU/mg.

Nidaamka falcelinta iyo xaaladaha

1. Isku darka qaddarka ku habboon ee Capped RNA iyo H2O-free RNase ee tuubbada microfuge 1.5 mL ilaa mugga kama dambaysta ah ee 16.0 µL.

2. Kuleyl 65 ℃ 5 daqiiqo oo ay ku xigto baraf-qubeyska 5 daqiiqo.

3. Ku dar qaybaha soo socda sida ay u kala horreeyaan (loogu talagalay methylation of Capped RNA

in ka yar 10

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

4. Ku duri at 37 ℃ 1 saac (2 saacadood oo ku-tiirsad ayaa lagula talinayaa jajabka bartilmaameedka ee ka yar 200 nt).

Codsiyada

Si loo wanaajiyo muujinta mRNA inta lagu guda jiro isku-dubarid-yar-yar iyo tijaabooyinka gudbinta.

Qoraalada isticmaalka

1. Kahor falcelinta, RNA waa in la nadiifiyaa oo lagu milmaa biyaha nucleus-ka, dhammaan xalalka waa in aysan ku jirin wax EDTA iyo ions.

2. Waxaa lagu talinayaa in lagu kululeeyo saamiga RNA at 65 ℃ 5 daqiiqo ka hor falcelinta si meesha looga saaro qaab-dhismeedka sare ee 5'dhamaadka qoraalka.Waxaa lagu kordhin karaa ilaa 10 daqiiqo qaab dhismeedka adag ee 5 ′-terminal.