BspQI
Enwere ike igosipụta BspQI n'otu n'otu na E. coli nke nwere ike ịmata saịtị ndị akọwapụtara ma mepụta n'okpuru
BspQI, ihe mgbochi IIs mmachi endonuclease restriction endonuclease, sitere na recombinant E. coli strain nke na-ebu BspQI gen cloned ma gbanwee site na Bacillus sphaericus.Ọ nwere ike ịmata saịtị ndị akọwapụtara, yana usoro amata na saịtị mkpochapụ bụ ndị a:
5' · · · · GCTCTTC(N) · · · · · · · · · · · 3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Njirimara ngwaahịa
1. Ọrụ dị elu, mgbaze ngwa ngwa;
2. Ọrụ kpakpando dị ala, na-eme ka ịkpụchasị mma dị ka "scalpel";
3. Enweghị BSA na anụmanụ sitere n'efu;
Mmetụta Methylation
Dna methylation:Ọ bụghị Mmetụta;
Dcm methylation:Ọ bụghị Mmetụta;
CpG Methylation:Ọ bụghị Mmetụta;
Ọnọdụ nchekwa
Ekwesịrị ibupu ngwaahịa ahụ ≤ 0℃;Na-echekwa na -25 ~-15 ℃ ọnọdụ.
Nchekwa nchekwa
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 na 50% glycerol (pH 7.0 @ 25°C).
Nkọwa nkeji
A kọwapụtara otu nkeji dị ka ọnụọgụ enzyme achọrọ iji gbarie 1µg nke DNA njikwa ime n'ime elekere 1 na 50 Celsius na mkpokọta mmeghachi omume nke 50 µL.
Njikwa ogo
Nnwale ịdị ọcha nke Protein (SDS-PAGE):A na-ekpebi ịdị ọcha nke BspQI ≥95% site na nyocha SDS-PAGE.
RNase:10U nke BspQI nwere 1.6μg MS2 RNA maka awa 4 na 50 ℃ anaghị eweta mbibi dị ka agarose gel electrophoresis siri kpebie.
Ọrụ DNA na-abụghị nke akọwapụtara:10U nke BspQI nwere 1μg λ DNA na 50℃ maka awa 16, ma e jiri ya tụnyere 50℃ maka 1hour, ọ dịghị amịpụta DNA gafere dị ka agarose gel electrophoresis kpebiri.
Njikọ na mwepu:Mgbe mgbaze nke 1 μg λDNA na 10U BspQI, enwere ike ijikọta iberibe DNA na ligase T4 DNA na 16ºC.Enwere ike iji BspQI gbutu iberibe irighiri ihe ndị a.
E. coli DNA: E.coli 16s rDNA-kpọmkwem TaqMan qPCR nchọpụta gosiri na E.coli genome residue ≤ 0.1pg/ul.
Ihe fọdụrụ na protein ndị ọbịa:≤50 ppm
Ọrịa nje Endotoxin: LAL-ule, dị ka Chinese Pharmacopoeia IV 2020 mbipụta, gel ókè ule usoro, izugbe iwu (1143).Ọdịnaya endotoxin nke nje bacteria kwesịrị ịbụ ≤10 EU/mg.
Usoro mmeghachi omume na ọnọdụ
| Akụkụ | Olu |
| BspQ I (10 U/μL) | 1 μl |
| DNA | 1 μg |
| 10 x BspQ I nchekwa | 5 μl |
| dd H2O | Ihe dị ka 50 μl |
Ọnọdụ mmeghachi omume: 50 ℃, 1 ~ 16 h.
Ịgbanyụ ọkụ: 80°C maka nkeji 20.
Usoro mmeghachi omume akwadoro na ọnọdụ nwere ike inye mmetụta mgbaze enzyme dịtụ mma, nke bụ naanị maka nkọwa, biko rụtụ aka na nsonaazụ nnwale maka nkọwa.
Ngwa ngwaahịa
Mmachibido mgbaze endonuclease, cloning ngwa ngwa.
Ihe ndetu
1. Olu nke enzyme ≤ 1/10 nke olu mmeghachi omume.
2. Ọrụ kpakpando nwere ike ime mgbe mkpokọta glycerol karịrị 5%.
3. Mmebi ihe nwere ike ime mgbe mkpụrụ n'okpuru oke akwadoro.
