Ajesara Iwoye Capping Enzyme
Enzymu capping virus Vaccinia jẹ lati inu igara E. coli ti o tun gbejade awọn jiini fun henensiamu capping Vaccinia.Enzymu ẹyọkan yii jẹ ti awọn ipin meji (D1 ati D12) ati pe o ni awọn iṣẹ enzymatic mẹta (RNA triphosphatase ati guanylyltransferase nipasẹ ipin D1 ati guanine methyltransferase nipasẹ apakan D12).Kokoro Vaccinia Capping Enzyme jẹ doko lati ṣe itara iṣelọpọ ti iṣeto fila, eyiti o le ni pataki so eto fila fila 7-methylguanylate (m7Gppp, Cap 0) si 5′ opin RNA.Eto fila (Fila 0) ṣe ipa pataki ninu imuduro mRNA, gbigbe ati itumọ ni awọn eukaryotes.Capping RNA nipasẹ iṣesi enzymatic jẹ ọna ti o munadoko ati irọrun eyiti o le mu iduroṣinṣin pọ si ati itumọ RNA fun transcription in vitro, gbigbe, ati microinjection.
Awọn eroja
Enzyme Capping Iwoye Ajesara (10 U/μL)
10× Capping Buffer
Awọn ipo ipamọ
-25～- 15℃ fun ibi ipamọ (Yago fun awọn iyipo didi-diẹ leralera)
Ifipamọ ipamọ
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Unit Definition
Ẹyọ kan ti ọlọjẹ Vaccinia Capping Enzyme jẹ asọye bi iye henensiamu ti o nilo lati ṣafikun 10pmol ti GTP sinu iwe-kikọ 80nt ni wakati kan ni 37°C.
Iṣakoso didara
Exonuclease:10U ti ọlọjẹ Vaccinia Capping Enzyme pẹlu 1μg λ-Hind III digest DNA ni 37 ℃ fun wakati 16 ko fa ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
Endonuclease:10U ti ọlọjẹ Vaccinia Capping Enzyme pẹlu 1μg λDNA ni 37℃ fun wakati 16 ko fa ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
Nickase:10U ti ọlọjẹ Vaccinia Capping Enzyme pẹlu 1 μg pBR322 ni 37 ℃ fun wakati 16 ko fa ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
RNase:10U ti ọlọjẹ Vaccinia Capping Enzyme pẹlu 1.6μg MS2 RNA fun awọn wakati 4 ni 37℃ ko fa ibajẹ bi a ti pinnu nipasẹ agarose gel electrophoresis.
1.coli DNA:10U ti ọlọjẹ Vaccinia Capping Enzyme jẹ iboju fun wiwa E. coli genomic DNA nipa lilo TaqMan qPCR pẹlu awọn alakoko kan pato fun E. coli 16S rRNA agbegbe.E. coli genomic DNA kontaminesonu jẹ≤1 E. coli genome.
2.Kokoro Endotoxin: LAL-igbeyewo, ni ibamu si 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. Ilana Capping (iwọn idahun: 20 μL)
Ilana yii wulo fun ifapa capping ti 10μg RNA (≥100 NT) ati pe o le ṣe iwọn ni ibamu si awọn ibeere idanwo.
I) Darapọ 10μg RNA ati H2O ti ko ni Nuclease ni tube microfuge milimita 1.5 si iwọn ipari ti 15.0 µL.* 10× Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Ooru ni 65 ℃ fun awọn iṣẹju 5 atẹle nipasẹ iwẹ yinyin fun iṣẹju 5.
3) Ṣafikun awọn paati wọnyi ni aṣẹ ti a pato
| Clagbara | Volomi |
| Denatured RNA (≤10μg, ipari≥100 NT) | 15 μL |
| 10×Afifififipamọ* | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Enzyme Capping Kokoro ajesara (10U/μL) | 1 μL |
*10× Capping Buffer:0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)
4) Incubate ni 37°C fun ọgbọn išẹju 30, RNA ti wa ni capped ati setan fun awọn ohun elo isalẹ.
2. 5′ ebute lebeli lenu (iwọn idahun: 20 μL)
Ilana yii jẹ apẹrẹ lati ṣe aami RNA ti o ni triphosphate 5 kan ati pe o le ṣe iwọn ni ibamu si awọn ibeere.Imuṣiṣẹ ti iṣakojọpọ aami yoo ni ipa nipasẹ ipin molar ti RNA: GTP, ati akoonu GTP ninu awọn ayẹwo RNA.
1) Darapọ iye ti o yẹ ti RNA ati H2O ti ko ni Nuclease ni tube microfuge milimita 1.5 si iwọn ipari ti 14.0 µL.
2) Ooru ni 65 ℃ fun awọn iṣẹju 5 atẹle nipasẹ iwẹ yinyin fun iṣẹju 5.
3) Ṣafikun awọn paati atẹle ni aṣẹ ti a pato.
| Clagbara | Volomi |
| Denatured RNA | 14 μL |
| 10× Capping Buffer | 2 μL |
| Apapo GTP** | 2 μL |
| SAM (2 mM) | 1 μL |
| Enzyme Capping Kokoro ajesara (10U/μL) | 1 μL |
** GTP MIX tọka si GTP ati nọmba kekere ti awọn asami.Fun ifọkansi ti GTP, tọka sisi Akọsilẹ 3.
4) Incubate ni 37°C fun ọgbọn išẹju 30, RNA 5′ opin ti wa ni aami ni bayi ati ṣetan fun isalẹ
Awọn ohun elo
1. Capping mRNA ṣaaju ṣiṣe awọn igbelewọn itumọ / in vitro itumọ
2. Ifi aami 5 'opin mRNA
Awọn akọsilẹ lori lilo
1.Alapapo ojutu ti RNA saju si abeabo pẹlu Vaccinia Capping Enzyme yọkuro igbekalẹ Atẹle lori 5'ipari ti kikowe naa.Fa akoko pọ si awọn iṣẹju 60 fun awọn iwe afọwọkọ pẹlu awọn opin 5'ends ti a mọ gaan.
2. RNA ti a lo fun awọn aati capping yẹ ki o di mimọ ṣaaju lilo ati daduro ni omi ti ko ni iparun.EDTA ko yẹ ki o wa ati ojutu yẹ ki o jẹ ofe ti iyọ.
3. Fun isamisi ipari 5′, ifọkansi GTP lapapọ yẹ ki o wa ni ayika 1-3 igba ifọkansi molar ti mRNA ninu iṣesi.
4. Awọn iwọn didun ti awọn lenu eto le ti wa ni ti iwọn soke tabi isalẹ gẹgẹ bi awọn gangan.
