DNase I
DNase I (Deoxyribonuclease I) ndi endodeoxyribonuclease yomwe imatha kugaya DNA ya zingwe ziwiri kapena ziwiri.Imazindikira ndikudula zomangira za phosphodiester kuti zipange monodeoxynucleotides kapena oligodeoxynucleotides imodzi kapena iwiri yokhala ndi magulu a phosphate pa 5'-terminal ndi hydroxyl pa 3'-terminal.Ntchito ya DNase I imadalira Ca2 + ndipo imatha kuyendetsedwa ndi ayoni achitsulo a divalent monga Mn2 + ndi Zn2 +.5mM Ca2+ imateteza enzyme ku hydrolysis.Mg2+ ikakhalapo, puloteniyo inkatha kuzindikira mwachisawawa ndi kuswa malo alionse pa chingwe chilichonse cha DNA.Pamaso pa Mn2+, zingwe ziwiri za DNA zimatha kuzindikirika nthawi imodzi ndikumangika pamalo omwewo kuti apange zidutswa za DNA kapena zidutswa zomata za DNA zomwe zili ndi 1-2 nucleotides.
Katundu Wazinthu
Bovine Pancreas DNase I adawonetsedwa mu mawonekedwe a yisiti ndikuyeretsedwa.
Cotsutsa
| Chigawo | Voliyumu | |||
| 0.1KU | 1 KU | 5KU | 50KU | |
| DNase I, RNase-free | 20μl pa | 200μL | 1ml pa | 10ml pa |
| 10 × DNase I Buffer | 1ml pa | 1ml pa | 5 × 1 mL | 5 × 10 mL |
Mayendedwe ndi Kusunga
1. Kukhazikika Kusunga: - 15 ℃~-25 ℃ posungira;
2.Transport Kukhazikika: Kuyenda pansi pa ayezi;
3. Amaperekedwa mu: 10 mM Tris-HCl, 2 mM CaCl2, 50% glycerol, pH 7.6 pa 25 ℃.
Tanthauzo la Chigawo
Gawo limodzi limatanthauzidwa ngati kuchuluka kwa enzyme yomwe ingawononge 1 µg ya pBR322 DNA mu mphindi 10 pa 37 ° C.
Kuwongolera Kwabwino
RNase:5U ya DNase I yokhala ndi 1.6 μg MS2 RNA kwa maola 4 pa 37 ℃ sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Bakiteriya Endotoxin:Kuyesa kwa LAL, malinga ndi kope la Chinese Pharmacopoeia IV 2020, njira yoyesera malire a gel, malamulo onse (1143).Mabakiteriya endotoxin ayenera kukhala ≤10 EU/mg.
Malangizo Ogwiritsa Ntchito
1.Konzekerani yankho mu chubu lopanda RNase molingana ndi zomwe zalembedwa pansipa:
| Chigawo | Voliyumu |
| RNA | X μg |
| 10 × DNase I Buffer | 1 ml |
| DNase I, RNase-free(5U/μL) | 1 U pa μg RNA |
| ddH2O | Mpaka 10 μL |
2.37 ℃ kwa mphindi 15;
3.Onjezani buffer yothetsa kuti muyimitse zomwe zikuchitika, ndi kutentha pa 65 ℃ kwa mphindi za 10 kuti mutsegule DNase I. Chitsanzocho chikhoza kugwiritsidwa ntchito mwachindunji poyesera zolembera.
Zolemba
1.Gwiritsani ntchito 1U DNase I pa μg ya RNA, kapena 1U DNase I pa zosakwana 1μg za RNA.
2.EDTA iyenera kuwonjezeredwa kumalo omaliza a 5 mM kuti ateteze RNA kuti isawonongeke panthawi ya enzyme inactivation.
