DNase I
I-DNase I (i-Deoxyribonuclease I) yi-endodeoxyribonuclease ekwazi ukwetyisa i-DNA enemisonto emibini.Iqaphela kwaye iqhawule iibhondi ze-phosphodiester ukuvelisa i-monodeoxynucleotides okanye i-oligodeoxynucleotides enye okanye kabini kunye namaqela e-phosphate kwi-5'-terminal kunye ne-hydroxyl kwi-3'-terminal.Umsebenzi we-DNase I ixhomekeke kwi-Ca2 + kwaye inokuthi isebenze ngee-ion zetsimbi ze-divalent ezifana ne-Mn2 + kunye ne-Zn2 +.I-5mM Ca2 + ikhusela i-enzyme kwi-hydrolysis.Xa kukho iMg2+, le enzyme inokubona ngokungenamkhethe ize icande kuyo nayiphi na indawo kuwo nawuphi na umcu weDNA.Phambi kobukho be-Mn2+, imisonto ephindwe kabini ye-DNA inokubonwa ngaxeshanye kwaye icandeke phantse kwindawo enye ukuze yenze amaqhekeza e-DNA esiphelo esicaba okanye amaqhekeza e-DNA ancamathelayo ane-1-2 ye-nucleotides ephumayo.
Ipropati yeMveliso
I-Bovine Pancreas DNase I yabonakaliswa kwinkqubo yokubonakalisa igwele kwaye yahlanjululwa.
Cabachasi
| Icandelo | Umthamo | |||
| 0.1KU | 1KU | 5KU | 50KU | |
| DNase I, RNase-free | 20μL | 200μL | 1mL | 10mL |
| 10×DNase I Buffer | 1mL | 1mL | 5×1mL | 5× 10mL |
Ezothutho kunye noGcino
1. Uzinzo loGcino: – 15℃~-25℃ yokugcina;
2.Uzinzo lwezoThutho: Uthutho phantsi kweepakethe zomkhenkce;
3. Inikwe: 10 mM Tris-HCl, 2 mM CaCl2, 50% glycerol, pH 7.6 ku-25℃.
Inkcazelo yeyunithi
Iyunithi enye ichazwa njengobungakanani be-enzayimi eya kuthoba ngokupheleleyo i-1 µg ye-pBR322 DNA kwimizuzu eli-10 ku-37°C.
Ulawulo lwemeko
RNase:I-5U ye-DNase I kunye ne-1.6 μg MS2 RNA kwiiyure ze-4 kwi-37 ℃ ayivelisi ukuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.
Intsholongwane I-Endotoxin:Uvavanyo lwe-LAL, ngokwe-Chinese Pharmacopoeia IV 2020 edition, indlela yokuvavanya umda we-gel, umthetho jikelele (1143).Umxholo we-endotoxin yebhaktheriya kufuneka ube ≤10 EU/mg.
Imiyalelo yokusetyenziswa
1.Lungisa isisombululo sokusabela kwityhubhu ye-RNase-free ngokomlinganiselo odweliswe ngezantsi:
| Icandelo | Umthamo |
| I-RNA | X μg |
| 10 × DNase I Buffer | 1 μL |
| I-DNase I, RNase-free(5U/μL) | I-1 U nge-μg RNA |
| ddH2O | Ukuya kuthi ga kwi-10 μL |
2.37 ℃ imizuzu eyi-15;
3.Yongeza i-buffer yokuphelisa ukumisa ukusabela, kunye nokufudumala kwi-65℃ imizuzu eyi-10 ukwenza i-DNase I ingasebenzi.
Amanqaku
1.Sebenzisa i-1U DNase I nge-μg ye-RNA, okanye i-1U DNase I ngaphantsi kwe-1μg ye-RNA.
I-2.EDTA kufuneka ifakwe kwi-concentration yokugqibela ye-5 mM ukukhusela i-RNA ekuthotyweni ngexesha lokungasebenzi kwe-enzyme.
