Fayraska Tallaalka Capping Enzyme

Insaymamka daboolaya fayraska tallaalku waxa uu ka yimid dib-u-habayn E. coli cadaadis ah oo sidda hiddo-wadaha loogu talagalay enzyme-ka daboolidda tallaalka.Enzyme-gani wuxuu ka kooban yahay laba qaybood (D1 iyo D12) wuxuuna leeyahay saddex hawlood oo enzymatic ah (RNA triphosphatase iyo guanylyltransferase ee qaybta D1 iyo guanine methyltransferase ee qaybta D12).Fayraska Vaccinia Capping Enzyme wuxuu wax ku ool u yahay inuu kiciyo sameynta qaab dhismeedka koofiyadda, kaas oo si gaar ah ugu dhejin kara qaab dhismeedka koofiyada 7-methylguanylate (m7Gppp, Cap 0) ilaa 5′ dhamaadka RNA.Qaab dhismeedka daboolka (Cap 0) wuxuu door muhiim ah ka ciyaaraa xasilinta mRNA, gaadiidka iyo tarjumaada eukaryotes.Xakamaynta RNA ee falcelinta enzymatic waa hab wax ku ool ah oo fudud kaas oo si weyn u wanaajin kara xasilloonida iyo tarjumaada RNA ee gudbinta vitro, gudbinta, iyo isku-dubbaridka yar-yar.

Qaybaha

Xakamaynta fayraska tallaalka Enzyme (10 U/μL)

10× Capping Buffer

Xaaladaha kaydinta

-25～- 15℃ si loo kaydiyo (iska ilaali wareegyada qaboojinta ee soo noqnoqda)

Kaydinta kaydinta

20 mM Tris-HCl (pH 8.0), 100 mM NaCl,

1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Qeexitaan Unug

Hal cutub oo ka mid ah fayraska Vaccinia Capping Enzyme waxaa lagu qeexaa cadadka insaymiska loo baahan yahay si loogu daro 10pmol ee GTP qoraal 80nt ah 1 saac oo ah 37°C.

Xakamaynta tayada

Exonuclease:10U ee fayraska Vaccinia Capping Enzyme oo leh 1μg λ-Hind III dheefshiidka DNA-da 37 ℃ 16 saacadood ma keenayso wax hoos u dhac ah sida lagu go'aamiyay agarose gel electrophoresis.

Endonuclease:10U of Vaccinia fayraska Capping Enzyme oo leh 1μg λDNA at 37℃ 16 saacadood ma keenayso hoos u dhac sida ay go'aamisay agarose gel electrophoresis.

Nickase:10U ee fayraska Vaccinia Capping Enzyme oo leh 1 μg pBR322 at 37 ℃ 16 saacadood ma keenayso hoos u dhac sida lagu go'aamiyay agarose gel electrophoresis.

RNase:10U ee fayraska Vaccinia Capping Enzyme oo leh 1.6μg MS2 RNA 4 saacadood oo ah 37 ℃ ma keenayso hoos u dhac sida ay go'aamisay agarose gel electrophoresis.

1.coli DNA:10U ee fayraska Vaccinia Capping Enzyme waxaa laga baaraa joogitaanka E. coli genomic DNA iyadoo la isticmaalayo TaqMan qPCR oo leh qalabyo u gaar ah E. coli 16S rRNA goobta.E. coli genomic DNA-da wasakhaysan waa≤1 E. coli genome.

2.Bakteeriya Endotoxin: LAL-tijaabo, marka loo eego daabacaadda Chinese Pharmacopoeia IV 2020, habka tijaabada xadka jel, qaanuunka guud (1143).Bakteeriyada endotoxin waa inay noqotaa ≤10 EU/mg.

Nidaamka falcelinta iyo xaaladaha

1. Hab-maamuuska Capping (mugga falcelinta: 20 μL)

Habkani waa mid lagu dabaqi karo falcelinta daboolka ee 10μg RNA (≥100 nt) waxaana lagu kordhin karaa iyadoo loo eegayo baahida tijaabada ah.

I) Isku dar 10μg RNA iyo H2O-free nuclease gudaha tuubo microfuge 1.5 ml ilaa mugga kama dambaysta ah ee 15.0 µL.* 10× Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)

2) Kuleyl at 65 ℃ 5 daqiiqo oo ay ku xigto baraf baraf ah 5 daqiiqo.

3) Ku dar qaybaha soo socda sida ay u kala horreeyaan

* 10× Capping Buffer: 0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)

4) Ku dhaji 37 ° C 30 daqiiqo, RNA hadda waa la daboolay oo diyaar u ah codsiyada hoose.

2.5′ terminal falcelinta calaamadaynta (mugga falcelinta: 20 μL)

Hab-maamuuska waxaa loogu talagalay in lagu calaamadiyo RNA ka kooban 5 'triphosphate' waxaana loo kordhin karaa iyadoo loo eegayo baahida.Waxtarka ku darista summada waxa saamayn doona saamiga molar ee RNA: GTP, iyo sidoo kale nuxurka GTP ee muunadaha RNA.

1) Isku darka qadarka saxda ah ee RNA iyo H2O-free nuclease ee tuubada microfuge 1.5 ml ilaa mugga kama dambaysta ah ee 14.0 µL.

2) Kuleyl at 65 ℃ 5 daqiiqo oo ay ku xigto baraf baraf ah 5 daqiiqo.

3)Ku dar qaybaha soo socda sida ay u kala horreeyaan.

** GTP MIX waxa loola jeedaa GTP iyo tiro yar oo calaamadeyaal ah.Si aad u fiirsato GTP, tixraacilaa note 3.

4) Ku dhaji 37°C 30 daqiiqo, dhamaadka RNA 5′ hadda waa la calaamadeeyay oo diyaar u ah dhanka hoose

Codsiyada

1. Cafiska mRNA ka hor tarjumaada turjumaada/in vitro

2. Ku calaamadinta 5′ dhamaadka mRNA

Qoraalada isticmaalka

1.Kuleylinta xalka RNA ka hor inta aan lagu tallaalin Enzyme Vaccinia Capping Enzyme waxay meesha ka saaraysaa qaab dhismeedka labaad ee 5'dhamaadka qoraalka.Kordhi wakhti ilaa 60 daqiiqo qoraalada leh 5'dhamaadka si heer sare ah loo habeeyay.

2. RNA ee loo isticmaalo dareen-celinta daboolka waa in la nadiifiyaa ka hor inta aan la isticmaalin oo lagu hakiyaa biyaha aan nukleuska lahayn.EDTA waa inaysan joogin, xalkuna waa inuu ahaadaa mid ka xor ah milix.

3. Si loo calaamadiyo dhamaadka 5', wadarta guud ee GTP-du waa inay ahaataa 1-3 jeer fiirsashada molar ee mRNA ee falcelinta.

4. Mugga habka falcelinta waa la kordhin karaa ama hoos ayaa loo dhigi karaa sida dhabta ah.