BspQI
I-BspQI inokuphinda ibonakaliswe kwi-E. coli ekwazi ukuqaphela iziza ezithile kwaye iveliswe phantsi
I-BspQI, i-IIs isithintelo se-endonuclease endonuclease, ithathwe kwi-recombinant E. coli strain ethwala uhlobo lwe-BspQI oludityanisiweyo lwaza lwalungiswa ukusuka kwi-Bacillus sphaericus.Iyakwazi ukuqaphela iisayithi ezithile, kunye nolandelelwano lokuqondwa kunye neziza zokucoca zimi ngolu hlobo lulandelayo:
5' · · · · GCTCTTC(N) · · · · · · · · · · · ·3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Iimpawu zeMveliso
1. Umsebenzi ophezulu, ukugaya ngokukhawuleza;
2. Umsebenzi weenkwenkwezi ezisezantsi, ukuqinisekisa ukusika ngokuchanekileyo njenge "scalpel";
3. Ngaphandle kwe-BSA kunye nesilwanyana-imvelaphi simahla;
I-Methylation Sensitivity
Dndine-methylation:Ayinavakalelo;
Dcm methylation:Ayinavakalelo;
CpG Methylation:Ayinavakalelo;
Iimeko zokugcina
Imveliso kufuneka ithunyelwe ≤ 0℃;Gcina kwi -25 ~ - 15 ℃ imeko.
Isithinteli sogcino
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 kunye ne-50% glycerol (pH 7.0 @ 25 ° C).
Inkcazelo yeyunithi
Iyunithi enye ichazwa njengobungakanani be-enzyme efunekayo ukugaya i-1µg yolawulo lwangaphakathi lwe-DNA kwiyure eyi-1 kwi-50 ° C kwivolumu yokusabela iyonke ye-50 µL.
Ulawulo lwemeko
Uvavanyo lweProtein Purity Assay (SDS-PAGE):Ukucoceka kwe-BspQI kwaba ≥95% kunqunywe nguhlalutyo lwe-SDS-PAGE.
RNase:I-10U ye-BspQI kunye ne-1.6μg MS2 RNA kwiiyure ze-4 kwi-50 ℃ ayivelisi ukuthotywa njengoko kunqunywe yi-agarose gel electrophoresis.
Umsebenzi we-DNase ongaQukwanga:I-10U ye-BspQI ene-1μg λ DNA kwi-50℃ kwiiyure ze-16, xa kuthelekiswa ne-50℃ ye-1hour, ayivelisi i-DNA engaphezulu njengoko kumiselwe yi-agarose gel electrophoresis.
I-Ligation kunye nokusika kwakhona:Emva kokugaya i-1 μg λDNA ene-10U BspQI, amaqhekeza e-DNA anokudityaniswa ne-T4 DNA ligase kwi-16ºC.Kwaye la maqhekeza adibeneyo anokuphinda anqunywe nge-BspQI.
E. coli I-DNA: E. coli 16s rDNA-specific TaqMan qPCR ubhaqo lubonise ukuba E.coli genome intsalela ≤ 0.1pg/ul.
Intsalela yeprotheyini yokusingatha:≤ 50 ppm
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.
Inkqubo yokusabela kunye neemeko
| Icandelo | Umthamo |
| I-BspQ I(10 U/μL) | 1 μL |
| DNA | 1 μg |
| 10 x BspQ I Buffer | 5 μL |
| dd H2O | Ukuya kuthi ga kwi-50 μL |
Iimeko zokusabela:50℃, 1 ~ 16 h.
Ukungasebenzi kobushushu: 80°C ngemizuzu engama-20.
Inkqubo yokusabela ecetyiswayo kunye neemeko zinokubonelela ngesiphumo esihle sokwetyisa i-enzyme, eyenzelwe ireferensi kuphela, nceda ujonge iziphumo zovavanyo ngeenkcukacha.
Isicelo semveliso
Isithintelo se-endonuclease digestion, iCloning eKhawulezayo.
Amanqaku
1. Umthamo we-enzyme ≤ 1/10 yomthamo wokuphendula.
2. Umsebenzi weenkwenkwezi unokwenzeka xa i-glycerol concentration ingaphezulu kwe-5%.
3. Umsebenzi wokucoca unokwenzeka xa i-Substrate ingaphantsi komlinganiselo ocetyiswayo.
