mRNA Cap2'-O-Methyltransferase

mRNA Cap 2' -O-methyltransferase inachokera ku mtundu wina wa E. coli womwe umanyamula jini ya katemera wa mRNA Cap 2 ´-O-Methyltransferase.Enzyme iyi imawonjezera gulu la methyl pa malo a 2'-O a nucleotide yoyamba yomwe ili pafupi ndi kapu ya kapu pa 5'mapeto a RNA. -0) chifukwa cha kapu- 1 kapangidwe.

Kapangidwe ka Cap1 kumatha kukulitsa kumasulira bwino, kuwongolera kufotokozera kwa mRNA muzoyeserera za transfection ndi microinjection.Iyi imafuna RNA makamaka yokhala ndi kapu ya m7GpppN ngati gawo lapansi.Sizingagwiritse ntchito RNA ndi pN, ppN, pppN kapena GpppN kumapeto kwa 5′.Capped RNA itha kukonzedwa kudzera mu in vitro transcript pogwiritsa ntchito cap analog kapena enzymatic caping pogwiritsa ntchito Vaccinia Capping Enzyme.

Zigawo

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

10 × Capping Reaction Buffer

Kusungirako

-25 ～- 15 ℃ posungira (Pewani kuzizira kobwerezabwereza)

Bafa yosungirako

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.

Tanthauzo la unit

Chigawo chimodzi chimatanthauzidwa ngati kuchuluka kwa enzyme yofunikira ku methylate 10 pmoles ya 80 nt capped RNA transcript mu ola la 1 pa 37 ° C.

Mayesero a khalidwe labwino

Exonuclease:50U ya mRNA Cap 2 ´ -O-Methyltransferase yokhala ndi 1μg λ-Hind III digest DNA pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.

Endonuclease: 50 U ya mRNA Cap 2 ´ -O-Methyltransferase yokhala ndi 1 μg λDNA pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.

Nickase: 50U ya mRNA Kapu 2 ´ -O-Methyltransferase yokhala ndi 1μg pBR322 pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.

RNase: 50U ya mRNA Cap 2 ´ -O-Methyltransferase yokhala ndi 1.6μg MS2 RNA kwa maola 4 pa 37 ℃ sichipereka kuwonongeka monga momwe agarose gel electrophoresis amachitira.

E. koli DNA: 50U ya mRNA Cap 2 ´ -O-Methyltransferase imawunikiridwa kuti ikhalepoE. koli DNA ya genomic pogwiritsa ntchito TaqMan qPCR yokhala ndi zoyambira zaE. koli 16S rRNA malo.TheE. koli Kuwonongeka kwa DNA ya genomic ndi =1E. koli genome.

Bakiteriya Endotoxin: LAL-mayeso, malinga ndi kope la Chinese Pharmacopoeia IV 2020, njira yoyesera malire a gel, malamulo onse (1143).Mabakiteriya endotoxin ayenera kukhala =10 EU/mg.

Dongosolo ndi zikhalidwe

1. Phatikizani kuchuluka koyenera kwa Capped RNA ndi RNase-free H2O mu chubu cha microfuge cha 1.5 mL mpaka voliyumu yomaliza ya 16.0 µL.

2. Tenthetsani pa 65 ℃ kwa mphindi zisanu ndikutsatiridwa ndi madzi oundana kwa mphindi zisanu.

3. Onjezani zigawo zotsatirazi mu dongosolo lomwe latchulidwa (kwa methylation ya Capped RNA

zosakwana 10

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

4. Yambani pa 37 ℃ kwa ola la 1 (maola a 2 a incubation akulimbikitsidwa pa chidutswa chandamale zosakwana 200 nt).

Mapulogalamu

Kupititsa patsogolo mawu a mRNA panthawi yoyeserera ma microinjection ndi transfection.

Zolemba pakugwiritsa ntchito

1. Musanachitepo kanthu, RNA iyenera kuyeretsedwa ndikusungunuka m'madzi opanda nuclease, njira zonse siziyenera kukhala ndi EDTA ndi ayoni.

2. Ndikoyenera kutenthetsa chitsanzo cha RNA pa 65 ℃ kwa 5 min musanayambe kuchitapo kanthu kuti muchotse dongosolo lachiwiri pa 5'mapeto a zolembazo.Itha kukulitsidwa mpaka mphindi 10 pamapangidwe ovuta a 5' -terminal.