mRNA Cap2'-O-Methyltransferase
mRNA Cap 2 ′-O-methyltransferase ti wa lati inu igara E. coli atunko ti o gbe jiini fun vaccinia mRNA Cap 2 ′-O-Methyltransferase.Enzymu yii ṣe afikun ẹgbẹ methyl kan ni ipo 2'-O ti nucleotide akọkọ ti o wa nitosi si ọna fila ni 5'ipari ti RNA. Enzymu naa nlo S-adenosylmethionine (SAM) gẹgẹbi oluranlọwọ methyl si methylate capped RNA (fila). -0) Abajade ni a fila- 1 be.
Ẹya Cap1 le ṣe alekun iṣẹ ṣiṣe itumọ, imudarasi ikosile ti mRNA ni gbigbe ati awọn adanwo microinjection. Enzymu yii ni pataki nilo RNA pẹlu fila m7GpppN bi sobusitireti.Ko le lo RNA pẹlu pN, ppN, pppN tabi GpppN ni opin 5′.Capped RNA le wa ni pese sile nipasẹ transcription in vitro nipa lilo fila afọwọṣe tabi nipasẹ enzymatic capping lilo Vaccinia Capping Enzyme.
Awọn eroja
mRNA Cap 2'-O-Methyltransferase (50U/μL)
10× Capping Reaction saarin
Ibi ipamọ
-25 ～- 15 ℃ fun ibi ipamọ (Yago fun awọn iyipo didi-diẹ leralera)
Ifipamọ ipamọ
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.
Itumọ ẹyọkan
Ẹyọ kan jẹ asọye bi iye henensiamu ti o nilo lati methylate 10 pmoles ti 80 nt capped RNA transcript ni wakati 1 ni 37°C.
Awọn idanwo iṣakoso didara
Exonuclease: 50U ti mRNA Cap 2 '-O-Methyltransferase pẹlu 1μg λ-Hind III digest DNA ni 37 ℃ fun wakati 16 ko ni ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
Endonuclease: 50 U ti mRNA Cap 2 '-O-Methyltransferase pẹlu 1 μg λDNA ni 37 ℃ fun wakati 16 ko ni ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
Nickase: 50U ti mRNA Cap 2 '-O-Methyltransferase pẹlu 1μg pBR322 ni 37 ℃ fun wakati 16 ko ni ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
RNase: 50U ti mRNA Cap 2 '-O-Methyltransferase pẹlu 1.6μg MS2 RNA fun wakati 4 ni 37 ℃ ko ni ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
E. koli DNA: 50U ti mRNA Cap 2 '-O-Methyltransferase ti wa ni iboju fun wiwa tiE. koli DNA genomic nipa lilo TaqMan qPCR pẹlu awọn alakoko kan pato funE. koli 16S rRNA ibi.AwọnE. koli Jiinidi DNA jẹ = 1E. koli jiini.
Kokoro Endotoxin: LAL-igbeyewo, gẹgẹ bi Chinese Pharmacopoeia IV 2020 àtúnse, jeli iye igbeyewo ọna, gbogboogbo ofin (1143).Akoonu endotoxin kokoro yẹ ki o jẹ =10 EU/mg.
Ifesi eto ati ipo
1. Darapọ iye ti o yẹ ti Capped RNA ati H2O ti ko ni RNase ni tube microfuge 1.5 milimita si iwọn ipari ti 16.0 µL.
2. Ooru ni 65 ℃ fun 5 iṣẹju atẹle nipa yinyin-wẹ fun 5 iṣẹju.
3. Ṣafikun awọn paati wọnyi ni aṣẹ ti a sọ pato (fun methylation ti Capped RNA
kere ju 10
| Ẹya ara ẹrọ | Iwọn didun |
| Denatured capped RNA | 16 μL |
| 10X Idaduro Idalẹnu Capping* | 2 μL |
| SAM (4 mM) | 1 μL |
| mRNA Cap 2'-O-Methyltransferase (50 U/μL) | 1 μL |
| ddH2O | Si 20 μl |
*10× Capping Reaction Buffer: 500 mM Tris-HCl(pH 8.0, 25℃), 50 mM KCl, 10 mM MgCl2, 10 mM DTT.
4. Incubate ni 37 ℃ fun 1 wakati (2 wakati ti abeabo ti wa ni niyanju fun awọn afojusun ajeku kere ju 200 nt).
Awọn ohun elo
Lati mu ikosile mRNA pọ si lakoko microinjection ati awọn adanwo gbigbe.
Awọn akọsilẹ lori lilo
1. Ṣaaju ki o to lenu, RNA yẹ ki o di mimọ ati tituka ni omi ti ko ni iparun, gbogbo awọn ojutu ko yẹ ki o ni eyikeyi EDTA ati awọn ions.
2. A ṣe iṣeduro lati gbona RNA ayẹwo ni 65 ℃ fun awọn iṣẹju 5 ṣaaju ifarabalẹ lati yọ eto-atẹle kuro lori 5'ipari ti igbasilẹ naa.O le faagun si iṣẹju 10 fun eka 5 ′-terminal be.
