mRNA Cap2'-O-Methyltransferase

ʻO ka mRNA Cap 2' -O-methyltransferase i loaʻa mai ka recombinant E. coli strain e lawe ana i ka gene no ka vaccinia mRNA Cap 2 '-O-Methyltransferase.Hoʻohui kēia enzyme i kahi pūʻulu methyl ma ke kūlana 2'-O o ka nucleotide mua e pili ana i ka hoʻolālā pāpale ma ka hopena 5' o ka RNA. Hoʻohana ka enzyme i ka S-adenosylmethionine (SAM) ma ke ʻano he mea hāʻawi methyl i ka methylate capped RNA (cap). -0) ka hopena i ka hoʻolālā cap-1.

Hiki i ka Cap1 structure ke hoʻonui i ka pono unuhi, hoʻomaikaʻi i ka hōʻike ʻana o ka mRNA i ka transfection a me nā hoʻokolohua microinjection. Pono kēia enzyme i ka RNA me kahi pāpale m7GpppN ma ke ʻano he substrate.ʻAʻole hiki iā ia ke hoʻohana i ka RNA me ka pN, ppN, pppN a i ʻole GpppN ma ka hopena 5'.Hiki ke hoʻomākaukau ʻia ka RNA i hoʻopaʻa ʻia ma o ka transcription in vitro me ka hoʻohana ʻana i ka analog cap a i ʻole ka hoʻopaʻa enzymatic me ka hoʻohana ʻana i ka Vaccinia Capping Enzyme.

Nā ʻāpana

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

10×Capping Reaction Buffer

Waihona

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

Paʻa mālama

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.

Wehewehe ʻāpana

Hoʻokahi ʻāpana i wehewehe ʻia ʻo ka nui o ka enzyme e koi ʻia e methylate 10 pmoles o 80 nt capped RNA transcript i 1 hola ma 37°C.

Nā hōʻike mana maikaʻi

Exonuclease: 50U o ka mRNA Cap 2 ´ -O-Methyltransferase me 1μg λ-Hind III digest DNA ma 37 ℃ no 16 mau hola ʻaʻole ia e hoʻohaʻahaʻa ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

Endonuclease: 50 U o mRNA Cap 2 ´ -O-Methyltransferase me 1 μg λDNA ma 37 ℃ no nā hola 16 ʻaʻole e hoʻohua ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

Nickase: 50U o mRNA Cap 2 ´ -O-Methyltransferase me 1μg pBR322 ma 37 ℃ no 16 mau hola ʻaʻole ia e hoʻoemi ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

RNase: 50U o ka mRNA Cap 2 ´ -O-Methyltransferase me 1.6μg MS2 RNA no 4 mau hola ma 37 ℃ ʻaʻole e hoʻoemi ʻia e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.

E. coli DNA: 50U o ka mRNA Cap 2 ´ -O-Methyltransferase ua paleia no ka hikiE. coli DNA genomic e hoʻohana ana i ka TaqMan qPCR me nā primers kikoʻī no kaE. coli 16S rRNA wahi.ʻO kaE. coli ʻO ka hoʻohaumia DNA genomic he =1E. coli genome.

Bakteri Endotoxin: LAL-ho'āʻo, e like me ka Chinese Pharmacopoeia IV 2020 paʻi, gel kaʻina hoʻāʻo palena, kānāwai maʻamau (1143).Pono ka maʻiʻo endotoxin bacteria = 10 EU/mg.

Pūnaehana pane a me nā kūlana

1. E hoʻohui i ka nui kūpono o Capped RNA a me RNase-free H2O i loko o kahi paipu microfuge 1.5 mL i ka leo hope o 16.0 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 (no ka methylation o Capped RNA

emi iho malalo o 10

*10× Hoʻopaʻa Paʻa Paʻa: 500 mM Tris-HCl(pH 8.0, 25 ℃), 50 mM KCl, 10 mM MgCl2 、 10 mM DTT.

4. Hoʻomoʻa i ka 37 ℃ no 1 hola (2 hola o ka hoʻoulu ʻana i manaʻo ʻia no ka ʻāpana pahuhopu ma lalo o 200 nt).

Nā noi

E hoʻomaikaʻi i ka hōʻike mRNA i ka wā o ka microinjection a me nā hoʻokolohua transfection.

Nā memo no ka hoʻohana ʻana

1. Ma mua o ka pane ʻana, pono e hoʻomaʻemaʻe ʻia ka RNA a hoʻoheheʻe ʻia i loko o ka wai nuclease-free, ʻaʻole pono e loaʻa i nā haʻina a pau kekahi EDTA a me nā ion.

2. Manaʻo ʻia e hoʻomaʻamaʻa i ka hāpana RNA ma 65 ℃ no 5 min ma mua o ka hoʻoneʻe ʻana i ka hana lua ma ka hopena 5' o ka palapala.Hiki ke hoʻonui ʻia i 10 min no ka hoʻolālā 5 ´ -terminal paʻakikī.