Vaccinia Virus Capping Enzyme
Vaccinia virus capping enzyme inotorwa kubva kune recombinant E. coli strain inotakura majini eVaccinia capping enzyme.Iyi enzyme imwe chete inoumbwa nezvikamu zviviri (D1 uye D12) uye ine mabasa matatu enzymatic (RNA triphosphatase uye guanylyltransferase neD1 subunit uye guanine methyltransferase neD12 subunit).Vaccinia virus Capping Enzyme inoshanda kukonzeresa kuumbwa kwechivharo chimiro, chinogona kunyatso batanidza iyo 7-methylguanylate cap chimiro (m7Gppp, Cap 0) kune iyo 5 ′ kupera kweRNA.Cap chimiro (Cap 0) inoita basa rakakosha muRNA kugadzikana, kutakura uye kududzira mueukaryotes.Capping RNA ne enzymatic reaction inzira inoshanda uye yakapusa inogona kuvandudza zvakanyanya kugadzikana uye dudziro yeRNA ye in vitro transcription, transfection, uye microinjection.
Zvikamu
Vaccinia Virus Capping Enzyme (10 U/μL)
10 × Capping Buffer
Kuchengetedza mamiriro
-25～- 15℃ yekuchengetedza (Dzivisa kudzokororwa kwechando-kunyorova kutenderera)
Storage buffer
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Chikamu Tsanangudzo
Imwe unit yeVaccinia virus Capping Enzyme inotsanangurwa sehuwandu hweenzyme inodiwa kuisa 10pmol yeGTP mune 80nt chinyorwa muawa imwe pa37°C.
Quality Control
Exonuclease:10U yeVaccinia virus Capping Enzyme ine 1μg λ-Hind III digest DNA pa37 ℃ kwemaawa gumi nematanhatu haibudisi kushatisa sekutsanangurwa kweagarose gel electrophoresis.
Endonuclease:10U yeVaccinia virus Capping Enzyme ine 1μg λDNA pa 37 ℃ kwemaawa gumi nematanhatu haibudisi kuderedzwa sekutsanangurwa neagarose gel electrophoresis.
Nickase:10U yeVaccinia virus Capping Enzyme ine 1 μg pBR322 pa37 ℃ kwemaawa gumi nematanhatu haibudisi kushatisa sekutsanangurwa kweagarose gel electrophoresis.
RNase:10U yeVaccinia virus Capping Enzyme ine 1.6μg MS2 RNA kwemaawa mana pa37℃ haibudisi kushatisa sekunotemwa neagarose gel electrophoresis.
1.coli DNA:10U yeVaccinia virus Capping Enzyme inoongororwa kuvapo kweE. coli genomic DNA pachishandiswa TaqMan qPCR ine maprimers chaiwo eE. coli 16S rRNA locus.Kusvibiswa kweE. coli genomic DNA is≤1 E. coli genome.
2.Utachiona Endotoxin: LAL-yedzo, maererano neChinese Pharmacopoeia IV 2020 edition, gel muganho bvunzo nzira, general mutemo (1143).Bacterial endotoxin content inofanira kuva ≤10 EU/mg.
Reaction system uye mamiriro
1. Capping Protocol (reaction volume: 20 μL)
Iyi nzira inoshanda kune capping reaction ye10μg RNA (≥100 nt) uye inogona kukwidziridzwa maererano nekuyedza zvinodiwa.
I) Batanidza 10μg RNA uye Nuclease-isina H2O mu 1.5 ml microfuge chubhu kusvika kuvhoriyamu yekupedzisira ye15.0 µL.*10×Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Kupisa pa65 ℃ kwemaminitsi mashanu uchiteverwa nechando kugeza kwemaminetsi mashanu.
3) Wedzera zvinotevera zvikamu muhurongwa hwakatsanangurwa
| Coponent | Volume |
| Denatured RNA (≤10μg, kureba≥100 nt) | 15 μL |
| 10×Capping Buffer* | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia virus 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) Chengetedza pa 37°C kwemaminetsi makumi matatu, RNA ikozvino yavharwa uye yakagadzirira kushandiswa kwepasi.
2. 5′ terminal labeling reaction (rection volume: 20 μL)
Iyi protocol yakagadzirirwa kunyora RNA ine 5' triphosphate uye inogona kukwidziridzwa zvinoenderana nezvinodiwa.Kushanda kweiyo label kubatanidzwa kuchakanganiswa ne molar ratio yeRNA: GTP, pamwe neiyo GTP yemukati mune RNA samples.
1) Batanidza huwandu hwakakodzera hweRNA neNuclease-isina H2O muchubhu ye 1.5 ml microfuge kusvika pavhoriyamu yekupedzisira ye14.0 µL.
2) Kupisa pa65 ℃ kwemaminitsi mashanu uchiteverwa nechando kugeza kwemaminetsi mashanu.
3) Wedzera zvinotevera zvikamu muhurongwa hwataurwa.
| Coponent | Volume |
| Denatured RNA | 14 μL |
| 10 × Capping Buffer | 2 μL |
| GTP musanganiswa** | 2 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 μL |
** GTP MIX inoreva GTP uye nhamba diki yemakaki.Kuti uwane huwandu hweGTP, tarisakune Note 3.
4) Chengetedza pa 37°C kwemaminetsi makumi matatu, RNA 5′ kupera kwakanyorwa uye kwagadzirira kudzika.
Applications
1. Capping mRNA isati yaitwa miyedzo yeshanduro/in vitro dudziro
2. Kunyora 5' kupera kwemRNA
Zvinyorwa zvekushandisa
1.Kupisa mhinduro yeRNA isati yaiswa neVaccinia Capping Enzyme inobvisa chechipiri chimiro pa5'magumo echinyorwa.Wedzera nguva kune 60 maminetsi ezvinyorwa ane anozivikanwa zvakanyanya kurongeka 5'ends.
2. RNA inoshandiswa pakuita capping inofanira kucheneswa isati yashandiswa uye yakamiswa mumvura isina nuclease.EDTA haifaniri kuvapo uye mhinduro yacho inofanira kunge isina munyu.
3. Pakunyora 5' magumo, huwandu hweGTP hunofanirwa kunge hwakapoteredza 1-3 nguva ye molar concentration ye mRNA mukuita.
4. Vhoriyamu yemaitiro ekuita inogona kukwidziridzwa kumusoro kana pasi zvichienderana neiyo chaiyo.
