Mtengo wa BspQI
BspQI ikhoza kufotokozedwanso mu E. coli yomwe imatha kuzindikira malo enieni ndipo amapangidwa pansi
BspQI, IIs restriction endonuclease restriction endonuclease, imachokera ku mtundu wina wa E. coli womwe umanyamula mtundu wa BspQI wopangidwa ndi kusinthidwa kuchokera ku Bacillus sphaericus.Ikhoza kuzindikira masamba enaake, ndipo kutsatizana kozindikirika ndi mawebusayiti ndi motere:
5' · · · · GCTTC(N) · · · · · · · · · · · 3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Zogulitsa Zamankhwala
1. High ntchito, Fast chimbudzi;
2. Ntchito yotsika ya nyenyezi, kuonetsetsa kudula kolondola ngati "scalpel";
3. Popanda BSA ndi nyama-chiyambi kwaulere;
Methylation Sensitivity
Dndi methylation:Osamvera;
Dcm methylation:Osamvera;
CpG Methylation:Osamvera;
Zosungirako
Zogulitsa ziyenera kutumizidwa ≤ 0 ℃;Sungani pa -25 ~ - 15 ℃ chikhalidwe.
Bafa yosungirako
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 ndi 50% glycerol (pH 7.0 @ 25°C).
Tanthauzo la Chigawo
Chigawo chimodzi chimatanthauzidwa ngati kuchuluka kwa enzyme yomwe imayenera kukumba 1µg ya Internal control DNA mu ola limodzi pa 50 ° C mu kuchuluka kwa 50 µL.
Kuwongolera Kwabwino
Protein Purity Assay (SDS-PAGE):Kuyera kwa BspQI kunali ≥95% yotsimikiziridwa ndi kusanthula kwa SDS-PAGE.
RNase:10U ya BspQI yokhala ndi 1.6μg MS2 RNA kwa maola 4 pa 50 ℃ sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Zochita Zosagwirizana ndi DNase:10U ya BspQI yokhala ndi 1μg λ DNA pa 50 ℃ kwa maola 16, poyerekeza ndi 50 ℃ kwa ola limodzi, sipereka DNA yochulukirapo monga momwe agarose gel electrophoresis amachitira.
Ligation ndi kudula:Pambuyo pogaya 1 μg λDNA ndi 10U BspQI, zidutswa za DNA zimatha kulumikizidwa ndi T4 DNA ligase pa 16ºC.Ndipo tizidutswa ta ligated izi zitha kudulidwanso ndi BspQI.
E. koli DNA: E. coli 16s rDNA-enieni TaqMan qPCR anapeza kuti E.coli genome zotsalira ≤ 0.1pg/ul.
Zotsalira zama protein:≤ 50 ppm
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.
Dongosolo ndi zikhalidwe
| Chigawo | Voliyumu |
| BspQ I(10 U/μL) | 1 ml |
| DNA | 1 mkh |
| 10 x BspQ I Buffer | 5 ml |
| ndi H2O | Mpaka 50 μL |
Zomwe zimachitika: 50 ℃, 1 ~ 16h.
Kutentha kosagwira ntchito: 80 ° C kwa mphindi 20.
The analimbikitsa kachitidwe dongosolo ndi mikhalidwe angapereke bwino enzyme chimbudzi zotsatira, amene ndi kutchula kokha, chonde onani zotsatira experimental kuti mwatsatanetsatane.
Kugwiritsa ntchito mankhwala
Kuletsa endonuclease chimbudzi, Rapid cloning.
Zolemba
1. Kuchuluka kwa enzyme ≤ 1/10 ya volume reaction.
2. Ntchito ya nyenyezi ikhoza kuchitika pamene glycerol ndende ndi yoposa 5%.
3. Ntchito yodutsa imatha kuchitika ngati gawo lapansi lili pansi pa chiyerekezo chomwe chikulimbikitsidwa.
