mRNA Cap2'-O-Methyltransferase
mRNA Cap 2´ -O-methyltransferase derivatum est ex recombinante E. coli cola quod gena fert pro vaccinia variant Cap 2 -O-Methyltransferase.Hoc enzyme addit coetus yl globi in 2´-O positionis primae nucleotide adiacentis structurae pilei ad 5´finem RNA. Enzyme utetur S-adenosylmethionine (SAM) ut methyl donatoris ad methylatum capped RNA (cap. -0) inde in cap- I structuram.
Fabrica Cap1 augere potest efficientiam translationis, expressionem variantis in transfection et experimentorum microiniectionis. Hoc enzyme specie postulat RNA cum pileo m7GpppN substrato.Non potest uti RNA cum pN, ppN, pppN vel GpppN in 5´ fine.Capped RNA via in vitro transcriptio parari potest utens cap analogo vel per enzymatic capping utens Vaccinia Capping Enzyme.
Components
mRNA Cap 2´-O-Methyltransferase (50U/μL)
X, Capping reactionem Buffer
Repono
-25 15℃ pro repono fuge crebris Frigidus CALEFACTO cycles.
Repono quiddam
20 mM Trit-HCl（pH 8.0，25℃), 100 mM NaCl, 1 mM DTT, 0. 1 mM EDTA, 0. 1% Triton X— 100， 50 glycerolum.
Unitas definition
Unitas una definitur quantum enzyme ad methylatum requiritur 10 pmoles 80 nt capped RNA transcript in hora 1 37°C.
Qualis imperium assays
Exonuclease:50U mRNA Cap 2 -O-Methyltransferasis cum 1μg λ-Hind III digestum DNA in 37 ℃ per 16 horas non cedit degradatio secundum agarosium gel electronicum determinatum.
Endonuclease: 50 U mRNA Cap 2 -O-Methyltransferasis cum 1 μg λDNA in 37℃ per 16 horas nullam cedit deformitatem sicut agarose gel electronicae determinatae.
Nickase: 50U variantis Cap 2 -O-Methyltransferasis cum 1μg pBR322 in 37 ℃ per 16 horas nullam cedit degradationem sicut agarose gel electronicae determinatae.
RNase: 50U variantis Cap 2 -O-Methyltransferase cum 1.6μg MS2 RNA per 4 horas ad 37℃ nullum cedit deformitatem sicut agarose gel electronicae determinatae.
E. coli DNA: 50U of variant Cap 2 -O-Methyltransferase obiectu praesentiaeE. coli genomic DNA usura TaqMan qPCR cum primers specifica proE. coli 16S rrnat locum.TheE. coli genomic DNA contagione est = 1E. coli genome.
Bacterial Endotoxin: LAL-test, secundum pharmacopoeam Sinensem IV 2020 editum, modum testium gel limitis, regulae generalis (1143).Contentum bacterial endotoxinum =10 EU/mg debet esse.
Reactionem ratio et conditiones
1. Copulam congruam RNA Capped et RNase liberorum H2O compone in tubo microfugii 1.5 mL ad volumen finale 16.0 µL.
2. Calefacere 65 pro 5 minutes, post glaciem-balneum pro 5 minutes.
3. Adde sequentia membra ordine determinato (pro methylatione Capped RNA
minus quam X "
| Component | Magnitudo |
| Denatured capped RNA | 16 μL |
| 10X Capping reactionem Buffer * | 2 μL |
| SAM (4 mM) | 1 μL |
| mRNA Cap 2´-O-Methyltransferase (50 U/μL) | 1 μL |
| ddH2O | Ad 20 μL |
*10× Reactio Capping Buffer : 500 mM Tris-HCl (pH 8.0, 25℃), 50 mM KCl, 10 mM MgCl2 、 10 mM DTT.
4. Incubare 37° horae 1(2 horae incubationis commendatur pro fragmento scopo minus quam 200 nt).
Applications
Ad emendandum mRNA expressionem per experimenta microinjectionem et transitum.
Notae in usu
1. Ante reactionem, RNA in aqua nucleaso libera et dilui debet, omnes solutiones EDTA et iones continere non debent.
2. Commendatur specimen RNA in 65℃ calefacere pro 5 min ante reactionem ad removendum structuram secundariam in 5´ fine transcripti.Posset extendi ad 10 min pro complexione 5´terminalis structurae.
