DNase I.
DNase I (Deoxyribonuclease I) ni endodeoxyribonuclease ishobora gusya ADN imwe cyangwa ebyiri.Iremera kandi ikomatanya imigozi ya fosifori kugirango itange monodeoxynucleotide cyangwa oligodeoxynucleotide imwe cyangwa ebyiri-ebyiri hamwe na fosifate kumatsinda ya 5′-terminal na hydroxyl kuri 3′-terminal.Igikorwa cya DNase I giterwa na Ca2 + kandi gishobora gukoreshwa na ion zingana nicyuma nka Mn2 + na Zn2 +.5mM Ca2 + irinda enzyme hydrolysis.Imbere ya Mg2 +, enzyme yashoboraga kumenya no guhitisha urubuga urwo arirwo rwose rwa ADN.Imbere ya Mn2 +, imigozi ibiri ya ADN irashobora kumenyekana icyarimwe kandi igafatirwa ahantu hamwe kugirango habeho ibice bya ADN bigororotse cyangwa ibice bya ADN bifatanye hamwe na nucleotide 1-2.
Umutungo wibicuruzwa
Bovine Pancreas DNase Nerekanwe muri sisitemu yo kwerekana imisemburo no kwezwa.
CIbigize
| Ibigize | Umubumbe | |||
| 0.1KU | 1KU | 5KU | 50KU | |
| DNase I, RNase-yubusa | 20μL | 200μL | 1mL | 10mL |
| 10 × DNase I Buffer | 1mL | 1mL | 5 × 1mL | 5 × 10mL |
Gutwara no Kubika
1. Ububiko buhamye: - 15 ℃ ~ -25 ℃ kubika;
2.Guhindura ibicuruzwa: Gutwara munsi yububiko bwa barafu;
3. Yatanzwe muri: 10 mM Tris-HCl, 2MM CaCl2, 50% glycerol, pH 7,6 kuri 25 ℃.
Igisobanuro
Igice kimwe gisobanurwa nkubunini bwa enzyme izatesha agaciro rwose 1 µg ya ADN ya pBR322 muminota 10 kuri 37 ° C.
Kugenzura ubuziranenge
RNase:5U ya DNase I hamwe na 1,6 μg MS2 RNA kumasaha 4 kuri 37 ℃ nta musaruro ugabanuka nkuko byagenwe na agarose gel electrophorei.
Indwara ya bagiteri Endotoxin:Ikizamini cya LAL, ukurikije Pharmacopoeia yo mu Bushinwa IV 2020, uburyo bwo gupima gel, uburyo rusange (1143).Indwara ya bagiteri igomba kuba ≤10 EU / mg.
Amabwiriza yo Gukoresha
1.Gutegura igisubizo cyibisubizo muri tube idafite RNase ukurikije ibipimo byavuzwe hepfo:
| Ibigize | Umubumbe |
| RNA | X μg |
| 10 × DNase I Buffer | 1 μL |
| DNase I, RNase-yubusa (5U / μL) | 1 U kuri μg RNA |
| ddH2O | Kugera kuri 10 μL |
2.37 ℃ mu minota 15;
3. Ongeraho buffer yo guhagarika kugirango uhagarike reaction, hanyuma ushushe kuri 65 ℃ muminota 10 kugirango udakora DNase I. Icyitegererezo kirashobora gukoreshwa muburyo butaziguye mugupima inyandiko ikurikira.
Inyandiko
1. Koresha 1U DNase I kuri μg ya RNA, cyangwa 1U DNase I kuri munsi ya 1μg ya RNA.
2.EDTA igomba kongerwamo ingufu za 5 mM kugirango irinde RNA kwangirika mugihe idakora enzyme.
