Vaccinia Virus Capping Enzyme

Loaʻa ʻia ka enzyme capping virus Vaccinia mai kahi hoʻohui E. coli strain e lawe i nā genes no ka Vaccinia capping enzyme.Hoʻokumu ʻia kēia enzyme hoʻokahi i ʻelua subunits (D1 a me D12) a he ʻekolu hana enzymatic (RNA triphosphatase a me guanylyltransferase e ka D1 subunit a me guanine methyltransferase e ka D12 subunit).ʻO ka Vaccinia virus Capping Enzyme ka mea e hiki ai ke hoʻopaʻa i ka hoʻokumu ʻia ʻana o ke ʻano o ka pāpale, hiki ke hoʻopili pono i ke ʻano o ka pāpale 7-methylguanylate (m7Gppp, Cap 0) i ka hopena 5′ o RNA.He kuleana koʻikoʻi ke ʻano o ka pāpale (Cap 0) i ka hoʻopaʻa ʻana o ka mRNA, ka lawe ʻana a me ka unuhi ʻana i nā eukaryotes.ʻO ka hoʻopaʻa ʻana i ka RNA ma ka hopena enzymatic he ala kūpono a maʻalahi hiki ke hoʻomaikaʻi nui i ka paʻa a me ka unuhi ʻana o RNA no ka transcription in vitro, transfection, a me microinjection.

Nā ʻāpana

Vaccinia Virus Capping Enzyme (10 U/μL)

10×Capping Buffer

Nā kūlana mālama

-25～- 15 ℃ no ka mālama ʻana （ Hōʻalo i nā pōʻai hoʻoheheʻe-hoʻohue hou)

Paʻa mālama

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

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

Wehewehe Unite

Hoʻokahi ʻāpana o Vaccinia virus Capping Enzyme i wehewehe ʻia ʻo ka nui o ka enzyme e pono ai e hoʻokomo i ka 10pmol o GTP i loko o kahi kope 80nt i ka hola 1 ma 37°C.

Ka Mana Mana

Exonuclease:ʻO 10U o Vaccinia virus Capping Enzyme me 1μg λ-Hind III digest DNA ma 37 ℃ no 16 mau hola ʻaʻole e hoʻohua ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

Endonuclease:ʻO 10U o Vaccinia virus Capping Enzyme me 1μg λDNA ma 37 ℃ no nā hola 16 ʻaʻole e hoʻohua ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

Nickase:ʻO 10U o Vaccinia virus Capping Enzyme me 1 μg pBR322 ma 37 ℃ no 16 mau hola ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

RNase:ʻO 10U o Vaccinia virus Capping Enzyme me 1.6μg MS2 RNA no 4 mau hola ma 37 ℃ ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

1.coli DNA:10U o Vaccinia virus Capping Enzyme ua hoʻopaʻa ʻia no ka loaʻa ʻana o E. coli genomic DNA me ka hoʻohana ʻana iā TaqMan qPCR me nā mea kikoʻī kikoʻī no ka E. coli 16S rRNA locus.ʻO ka E. coli genomic DNA contamination is≤1 E. coli genome.

2.Bakteri Endotoxin: LAL-hōʻike, e like me ka Chinese Pharmacopoeia IV 2020 edition, gel limit test method, general rule (1143).Pono ka maʻiʻo endotoxin bacteria ≤10 EU/mg.

Pūnaehana pane a me nā kūlana

1. Ka Hoʻopiʻi Kūʻai (ka nui o ka pane: 20 μL)

Pili kēia kaʻina hana i ka hopena capping o 10μg RNA (≥100 nt) a hiki ke hoʻonui ʻia e like me nā koi hoʻokolohua.

I) Hoʻohui i ka 10μg RNA a me ka Nuclease-free H2O i loko o kahi pahu microfuge 1.5 ml a hiki i ka leo hope o 15.0 µL.*10×Ka Paʻa Paʻa: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)

2) E wela ma 65 ℃ no 5 mau minuke a me ka ʻauʻau hau no 5 mau minuke.

3) E hoʻohui i nā ʻāpana i lalo i ke kauoha i kuhikuhi ʻia

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

4) Hoʻomoʻa ma 37 ° C no 30 mau minuke, ua hoʻopaʻa ʻia ʻo RNA a mākaukau no nā noi i lalo.

2. 5′ pahu hoʻopaʻa inoa ʻana (ka nui pane: 20 μL)

Hoʻolālā ʻia kēia protocol e lepili i ka RNA i loaʻa kahi 5' triphosphate a hiki ke hoʻonui ʻia e like me nā koi.Hoʻopili ʻia ka maikaʻi o ka hoʻohui ʻana i ka lepili e ka ratio molar o RNA: GTP, a me ka ʻike GTP i nā laʻana RNA.

1) Hoʻohui i ka nui kūpono o ka RNA a me ka Nuclease-free H2O i loko o kahi paipu microfuge 1.5 ml i ka leo hope o 14.0 µL.

2) E wela ma 65 ℃ no 5 mau minuke a me ka ʻauʻau hau no 5 mau minuke.

3) E hoʻohui i kēia mau ʻāpana i ke kauoha i kuhikuhi ʻia.

** ʻO GTP MIX e pili ana iā GTP a me kahi helu liʻiliʻi o nā māka.No ka noʻonoʻo ʻana o GTP, e nānāi ka Nānā 3.

4) Hoʻomoʻa ma 37°C no 30 mau minuke, ua hōʻailona ʻia ka hopena RNA 5′ a mākaukau no ka lalo.

Nā noi

1. Hoʻopaʻa ʻia ka mRNA ma mua o ka hoʻāʻo ʻana i ka unuhi/unuhi in vitro

2. Lepili 5' hope o ka mRNA

Nā memo no ka hoʻohana ʻana

1.ʻO ka hoʻomaʻamaʻa ʻana i ka hopena o RNA ma mua o ka hoʻoulu ʻana me ka Vaccinia Capping Enzyme e hoʻoneʻe i ka hana lua ma ka hopena 5' o ka transcript.E hoʻolōʻihi i ka manawa i 60 mau minuke no nā transcripts me nā 5'ends i ʻike ʻia.

2. Pono e ho'oma'ema'e 'ia ka RNA i ho'ohana 'ia no ka ho'opili 'ana ma mua o ka ho'ohana 'ana a ho'oku'u 'ia i ka wai nuclease-free.ʻAʻole pono ka EDTA a ʻaʻohe paʻakai ka hopena.

3. No ka ho'opa'a 'ana i ka hopena 5', 'o ka nui o ka mana'o GTP ma kahi o 1-3 manawa o ka mola o ka mRNA ma ka pane.

4. Hiki ke hoʻonui a hoʻohaʻahaʻa i ka nui o ka ʻōnaehana pane e like me ka mea maoli.