Vaccinia Virus Capping Enzyme
Vaccinia virus capping enzyme derivatur a recombinante E. coli cola gena defert enzyme pro Vaccinia capping.Haec enzyma una ex duobus subunitis (D1 et D12) componitur et tres enzymaticas actiones habet (RNA triphosphatase et guanylyltransferasis per subunit et guaninam methyltransferas D1 per subunit D12).Virus Vaccinia Capping Enzyme efficax est ad conformationem structurae pilei catalyzae, quae specie 7-methylguanylatae structurae pilei (m7Gppp, Cap 0) ad 5′ finem RNA apponere potest.Cap structura (Cap 0) magni ponderis munus agit in variantibus stabilizationibus, translationibus et translationibus in eukaryotes.Capping RNA per reactionem enzymaticam methodus efficax et simplex est, quae signanter stabilitatem et translationem RNA emendare potest in vitro transcriptione, transfectio, et microinjectione.
Components
Vaccinia Virus Capping Enzyme (10 U/μL)
10×Capping Buffer
Repono conditionibus
-25～- 15℃ pro repono fuge crebris Frigidus CALEFACTO cycles.
Repono quiddam
20 mM Tris-HCl (pH 8.0), 100 mM NaCl
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- C, L% glycerolum.
Unitas Definition
Vna Vacciniae virii Capping Enzyme unitas definitur enzyme quantitatem 10pmol GTP in transcripto 80ms in hora 1 37°C incorporandi.
Regimen quālitātis
Exonuclease:10U Vacciniae virus Capping Enzyme cum 1μg λ-Hind III digestum DNA in 37 ℃ per 16 horas nullam degradationem cedit sicut agarose gel electronicae determinatae.
Endonuclease:10U Vacciniarum virus Capping Enzyme cum 1μg λDNA in 37℃ per 16 horas nullam degradationem cedit sicut agarose gel electronicae determinatae.
Nickase:10U Vacciniae virus Capping Enzyme cum 1 μg pBR322 ad 37 per 16 horas nullum degradationem cedit sicut agarose gel electronicae determinatae.
RNAse:10U Vacciniae virus Capping Enzyme cum 1.6μg MS2 RNA per 4 horas ad 37℃ nullam degradationem reddit sicut agarose gel electronicae determinatae.
1.coli DNA:10U Vacciniae virus Capping Enzyme obiectu ad conspectum E. coli genomic DNA utens TaqMan qPCR cum primers specificis pro E. coli 16S rRNA locus.E. coli genomicum DNA contagione is≤1 E. coli genome.
2.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. Protocollum Capping (volumen reactionis: 20 μL)
Haec ratio applicabilis est ad reactionem capping de 10μg RNA (≥100 nt) et secundum exigentias experimentales potest ascendere.
I) Misce 10μg RNA et nucleas H2O in tubo microfugii 1.5 ml usque ad ultimum volumen 15.0 µL.*10× Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Calefacere 65℃ pro 5 minutes post glaciem balneum pro 5 minutes.
III) haec addere components in ordine certa
| Component | Volume |
| Denatured RNA (≤10μg, length≥100 nt) | 15 μL |
| 10× Capping Buffer * | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Virus Vaccinia Capping Enzyme (10U/μL) | 1 μL |
*10× Capping Buffer：0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)
4) Incubare 37°C pro 30 minutis, RNA nunc capped et ad applicationes amni paratas.
2. 5′ terminal labeling reactionem (Reactio volumen: 20 μL)
Hoc protocollum ordinatur ad pittacium RNA continens 5´ triphosphatem et secundum exigentias escendere potest.Efficax incorporationis pittacii a ratione molares RNA: GTP impacta est, necnon GTP contenta in RNA samples.
1) Copiam RNA et nuclei liberae H2O compone in tubo microfugio 1.5 ml usque ad ultimum volumen 14.0 µL.
2) Calefacere 65 pro 5 minutes post glaciem balneum pro 5 minutes.
3) Adde sequentia membra in ordinem datum est.
| Component | Volume |
| Denatured RNA | 14 μL |
| 10×Capping Buffer | 2 μL |
| GTP misce** | 2 μL |
| SAM (2 mM) | 1 μL |
| Virus Vaccinia Capping Enzyme (10U/μL) | 1 μL |
** GTP MIX refert ad GTP et parvum numerum venalicium.Ad intentionem GTP, referad Nota III.
4) Incubare 37°C pro 30 minutis, RNA 5′, finis nunc intitulatus et amni paratus
Applications
1. Capping mRNA prior translatione pertentat/in vitro translation
2. Labeling 5´ fine variant
Notae in usu
1.Solutionem RNA calefaciens ante incubationem cum Vaccinia Capping Enzyme structuram secundariam in 5´ fine transcripti removet.Extende tempus ad 60 minuta pro transcriptis cum notis valde structis 5´ finibus.
2. RNA ad motus cappingendos adhibita purgari debet ante usum et suspensum in aqua nucleaseo libera.EDTA adesse non debet, et solutio salium immunis sit.
3. Ad pter 5´ finem, intentio totalis GTP circa 1-3 tempora debet esse concentratio molaris variantis in reactione.
4. Volumen systematis reactionis secundum actualem sursum vel deorsum ascendere potest.
