mRNA Cap2'-O-Methyltransferase
mRNA Cap 2' -O-methyltransferase yakabva kune recombinant E. coli strain inotakura jena rekudzivirira mRNA Cap 2 ´-O-Methyltransferase.Iyi enzyme inowedzera boka re methyl panzvimbo ye 2'-O yekutanga nucleotide iri pedyo nekapu yakagadzirwa pa 5'kuguma kweRNA.Iyo enzyme inoshandisa S-adenosylmethionine (SAM) se methyl donor kune methylate capped RNA (cap. -0) zvichikonzera chivharo- 1 chimiro.
Chimiro cheCap1 chinogona kuwedzera kushandiswa kweshanduro, kuvandudza kutaura kwemRNA mukushandurwa uye kuedza kwe microinjection.Iyi enzyme inoda zvakananga RNA ine m7GpppN cap se substrate.Haikwanise kushandisa RNA ine pN, ppN, pppN kana GpppN pa5' yekupedzisira.Capped RNA inogona kugadzirwa kuburikidza ne in vitro transcript uchishandisa cap analog kana kuburikidza enzymatic capping uchishandisa iyo Vaccinia Capping Enzyme.
Zvikamu
mRNA Cap 2'-O-Methyltransferase (50U/μL)
10 × Capping Reaction Buffer
Storage
-25 ～- 15 ℃ yekuchengetedza (Dzivisa kudzokororwa kwechando-kunyorova kutenderera)
Storage buffer
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.
Tsanangudzo yeyuniti
Chimwe chikwata chinotsanangurwa sehuwandu hwe enzyme inodiwa ku methylate 10 pmoles ye80 nt capped RNA transcript muawa imwe pa37 ° C.
Kuongorora kwehutano
Exonuclease:50U ye mRNA Cap 2 ´ -O-Methyltransferase ine 1μg λ-Hind III digest DNA pa 37 ℃ kwemaawa gumi nematanhatu haibudisi kuderedzwa sekutsanangurwa neagarose gel electrophoresis.
Endonuclease: 50 U ye mRNA Cap 2 ´ -O-Methyltransferase ine 1 μg λDNA pa 37℃ kwemaawa 16 haibudisi kuora sezvinotsanangurwa neagarose gel electrophoresis.
Nickase: 50U ye mRNA Cap 2 ´ -O-Methyltransferase ine 1μg pBR322 pa 37 ℃ kwemaawa 16 haibudisi kuora sezvinotsanangurwa neagarose gel electrophoresis.
RNase: 50U ye mRNA Cap 2 ´ -O-Methyltransferase ine 1.6μg MS2 RNA kwemaawa mana pa 37 ℃ haibudisi kuora sezvinotsanangurwa neagarose gel electrophoresis.
E. coli DNA: 50U yemRNA Cap 2 ´ -O-Methyltransferase inoongororwa kuvapo kweE. coli genomic DNA uchishandisa TaqMan qPCR ine primers yakanangana neiyoE. coli 16S rRNA nzvimbo.TheE. coli genomic DNA kusvibiswa iri =1E. coli genome.
Utachiona Endotoxin: LAL-yedzo, maererano neChinese Pharmacopoeia IV 2020 edition, gel muganho bvunzo nzira, general mutemo (1143).Bacterial endotoxin content inofanira kuva =10 EU/mg.
Reaction system uye mamiriro
1. Sanganisa huwandu hwakakodzera hweCapped RNA uye RNase-isina H2O muchubhu ye 1.5 mL microfuge kusvika kuhuwandu hwekupedzisira hwe16.0 µL.
2. Kupisa pa 65℃ kwemaminitsi mashanu uchiteverwa nechando-kugeza kwemaminitsi mashanu.
3. Wedzera zvinotevera zvikamu muhurongwa hwakatarwa (yemethylation yeCapped RNA
isingasviki 10
| Chikamu | Volume |
| Denatured cappped RNA | 16 μL |
| 10X Capping Reaction Buffer* | 2 μL |
| SAM (4 mM) | 1 μL |
| mRNA Cap 2'-O-Methyltransferase (50 U/μL) | 1 μL |
| ddH2O | Kusvika ku20 μL |
*10× Capping Reaction Buffer : 500 mM Tris-HCl(pH 8.0, 25℃), 50 mM KCl, 10 mM MgCl2 、 10 mM DTT.
4. Isai pa 37 ℃ kweawa 1 (2 maawa ekuputira anokurudzirwa kune chinangwa chechidimbu chisingasviki 200 nt).
Applications
Kuvandudza mRNA kutaura panguva ye microinjection uye transfection kuyedza.
Zvinyorwa zvekushandisa
1. Pamberi pekuita, RNA inofanira kucheneswa uye yakanyungudutswa mumvura isina nuclease, zvose zvinogadzirisa hazvifaniri kunge zvine chero EDTA uye ions.
2. Inokurudzirwa kupisa sampuli yeRNA pa 65 ℃ ye 5 min isati yaita kuti ibvise chimiro chechipiri pa 5'kuguma kwechinyorwa.Inogona kuwedzerwa kusvika ku10 min kune yakaoma 5 ´ -terminal chimiro.
