Vaccinia Virus Capping Enzyme
Vaccinia virus capping enzyme yog muab los ntawm ib tug recombinant E. coli strain nqa cov noob rau Vaccinia capping enzyme.Qhov no ib leeg enzyme yog tsim los ntawm ob subunits (D1 thiab D12) thiab muaj peb enzymatic kev ua ub no (RNA triphosphatase thiab guanylyltransferase los ntawm D1 subunit thiab guanine methyltransferase los ntawm D12 subunit).Vaccinia virus Capping Enzyme yog qhov ua tau zoo rau kev tsim cov qauv cap, uas tuaj yeem tshwj xeeb xa mus rau 7-methylguanylate cap qauv (m7Gppp, Cap 0) mus rau 5' kawg ntawm RNA.Cap qauv (Cap 0) plays lub luag haujlwm tseem ceeb hauv mRNA stabilization, thauj thiab txhais lus hauv eukaryotes.Capping RNA los ntawm cov tshuaj tiv thaiv enzymatic yog ib txoj hauv kev zoo thiab yooj yim uas tuaj yeem txhim kho kev ruaj ntseg thiab kev txhais lus ntawm RNA rau hauv vitro transcription, transfection, thiab microinjection.
Cheebtsam
Vaccinia Virus Capping Enzyme (10 U/μL)
10 × Capping Buffer
Cia tej yam kev mob
-25 ~ 15 ℃ rau cia (Tsis txhob rov qab khov-thaw mus)
Cia tsis
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Unit txhais
Ib chav tsev ntawm Vaccinia virus Capping Enzyme yog txhais tau tias yog tus nqi ntawm cov enzyme uas yuav tsum tau koom nrog 10pmol ntawm GTP rau hauv 80nt cov ntawv teev lus hauv 1 teev ntawm 37 ° C.
Kev Tswj Xyuas Zoo
Exonuclease:10U ntawm Vaccinia virus Capping Enzyme nrog 1μg λ-Hind III zom DNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
Endonuclease:10U ntawm Vaccinia virus Capping Enzyme nrog 1μg λDNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
Nickase:10U ntawm Vaccinia virus Capping Enzyme nrog 1 μg pBR322 ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
RNase:10U ntawm Vaccinia virus Capping Enzyme nrog 1.6μg MS2 RNA rau 4 teev ntawm 37 ℃ yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
1.coli DNA:10U ntawm Vaccinia virus Capping Enzyme yog tshuaj xyuas qhov muaj E. coli genomic DNA siv TaqMan qPCR nrog primers tshwj xeeb rau E. coli 16S rRNA locus.E. coli genomic DNA paug yog ≤1 E. coli genome.
2.Kab mob Endotoxin: LAL-test, raws li Suav Pharmacopoeia IV 2020 ib tsab, gel limit test method, general rule (1143).Cov kab mob endotoxin cov ntsiab lus yuav tsum yog ≤10 EU / mg.
Cov tshuaj tiv thaiv kab mob thiab cov xwm txheej
1. Capping raws tu qauv (thov ntim: 20 μL)
Cov txheej txheem no muaj feem xyuam rau cov tshuaj tiv thaiv capping ntawm 10μg RNA (≥100 nt) thiab nws tuaj yeem ntsuas tau raws li qhov xav tau ntawm kev sim.
I) Muab 10μg RNA thiab Nuclease-free H2O hauv 1.5 ml microfuge raj rau qhov kawg ntim ntawm 15.0 µL.* 10 × Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH 8.0)
2) Tshav kub ntawm 65 ℃ rau 5 feeb tom qab da dej dej khov rau 5 feeb.
3) Ntxiv cov khoom nram qab no hauv qhov kev txiav txim tau teev tseg
| Component | Volum |
| Denatured RNA (≤10μg, ntev ≥100 nt) | 15 mL |
| 10 × Capping Buffer * | 2l miv |
| GTP (10 mM) | 1 mL ib |
| SAM (2 mM) | 1 mL ib |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 mL ib |
* 10 × Capping Buffer: 0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH8.0)
4) Ua kom sov ntawm 37 ° C rau 30 feeb, RNA tam sim no tau capped thiab npaj rau kev siv qis qis.
2. 5' Terminal UAS daim ntawv lo tshuaj tiv thaiv (react volume: 20 μL)
Cov txheej txheem no yog tsim los sau npe RNA uas muaj 5' triphosphate thiab nws tuaj yeem ntsuas tau raws li qhov xav tau.Kev ua tau zoo ntawm kev koom ua ke ntawm daim ntawv lo yuav cuam tshuam los ntawm molar piv ntawm RNA: GTP, nrog rau GTP cov ntsiab lus hauv RNA cov qauv.
1) Muab cov nqi tsim nyog ntawm RNA thiab Nuclease-free H2O hauv 1.5 ml microfuge raj rau qhov kawg ntim ntawm 14.0 µL.
2) Tshav kub ntawm 65 ℃ rau 5 feeb tom qab da dej dej khov rau 5 feeb.
3) Ntxiv cov khoom nram qab no hauv qhov kev txiav txim tau teev tseg.
| Component | Volum |
| Denatured RNA | 14l ua |
| 10 × Capping Buffer | 2l miv |
| GTP mix** | 2l miv |
| SAM (2 mM) | 1 mL ib |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 mL ib |
** GTP MIX hais txog GTP thiab cov cim me me.Rau qhov concentration ntawm GTP, xa musrau Note3.
4) Incubate ntawm 37 ° C rau 30 feeb, RNA 5' kawg yog tam sim no sau npe thiab npaj rau nqes hav
Daim ntawv thov
1. Capping mRNA ua ntej kev ntsuam xyuas kev txhais lus/hauv vitro txhais lus
2. Labeling 5' kawg ntawm mRNA
Cov lus qhia txog kev siv
1.Cua sov cov kev daws teeb meem ntawm RNA ua ntej incubation nrog Vaccinia Capping Enzyme tshem tawm cov qauv thib ob ntawm 5' kawg ntawm daim ntawv sau tseg.Txuas lub sijhawm mus rau 60 feeb rau cov ntawv sau tseg uas paub zoo heev 5'ends.
2. RNA siv rau capping cov tshuaj tiv thaiv yuav tsum tau ua kom huv si ua ntej siv thiab tshem tawm hauv cov dej tsis muaj nuclease.EDTA yuav tsum tsis txhob muaj thiab cov tshuaj yuav tsum tsis muaj ntsev.
3. Rau kev sau npe ntawm 5' kawg, tag nrho GTP concentration yuav tsum nyob ib ncig ntawm 1-3 npaug ntawm molar concentration ntawm mRNA hauv cov tshuaj tiv thaiv.
4. Lub ntim ntawm cov tshuaj tiv thaiv kab mob tuaj yeem nce lossis nqis raws li qhov tseeb.
