BspQI
BspQI e ka boela ea hlalosoa ka E. coli e ka tsebang libaka tse itseng 'me e hlahisoa ka tlas'a
BspQI, e leng IIs restriction endonuclease restriction endonuclease, e nkiloe ho mofuta oa recombinant E. coli o jereng mofuta oa BspQI o hlophisitsoeng le o fetotsoeng ho tsoa ho Bacillus sphaericus.E ka lemoha libaka tse ikhethileng, 'me tatellano ea tatellano le liwebsaete tsa cleavage ke tse latelang:
5' · · · ·GCTTC(N) · · · · · · · · · · · ·3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Likarolo tsa Sehlahisoa
1. Mosebetsi o phahameng, Ho potlakisa tšilo ea lijo;
2. Ts'ebetso ea linaleli tse tlase, ho netefatsa ho itšeha ka nepo joalo ka "scalpel";
3. Ntle le BSA le phoofolo-tšimoloho mahala;
Methylation Sensitivity
Dke methylation:Ha e Sensitive;
Dcm methylation:Ha e Sensitive;
CpG Methylation:Ha e Sensitive;
Maemo a polokelo
Sehlahisoa se lokela ho romelloa ≤ 0℃;Boloka ho -25 ~ - 15 ℃ boemo.
Sebaka sa polokelo
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 le 50% glycerol (pH 7.0 @ 25°C).
Tlhaloso ea Yuniti
Yuniti e le 'ngoe e hlalosoa e le palo ea enzyme e hlokahalang ho cheka 1µg ea DNA ea taolo ea ka hare ka hora e le 1 ho 50 ° C ka kakaretso ea karabelo ea 50 µL.
Taolo ea Boleng
Protein Purity Assay (SDS-PAGE):Bohloeki ba BspQI e ne e le ≥95% e khethiloeng ke tlhahlobo ea SDS-PAGE.
RNase:10U ea BspQI e nang le 1.6μg MS2 RNA bakeng sa lihora tse 4 ho 50 ℃ ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
Ts'ebetso e sa Totobalang ea DNase:10U ea BspQI e nang le 1μg λ DNA ho 50 ℃ bakeng sa lihora tse 16, ha e bapisoa le 50 ℃ bakeng sa 1hour, ha e fane ka DNA e feteletseng joalokaha e laetsoe ke agarose gel electrophoresis.
Ligation le ho khaola:Kamora tšilo ea 1 μg λDNA e nang le 10U BspQI, likhechana tsa DNA li ka hokahana le T4 DNA ligase ho 16ºC.'Me likhechana tsena tsa ligated li ka khaoloa hape ka BspQI.
E. coli DNA: Tlhahlobo ea E. coli 16s rDNA-specific TaqMan qPCR e bontšitse hore masalla a genome ea E.coli ≤ 0.1pg/ul.
Mesaletsa ea protheine e amohelang:≤ 50 ppm
Baktheria Endotoxin: Tlhahlobo ea LAL, ho latela khatiso ea China Pharmacopoeia IV 2020, mokhoa oa tlhahlobo ea moeli oa gel, molao o akaretsang (1143).Likahare tsa endotoxin ea baktheria e lokela ho ba ≤10 EU/mg.
Mokhoa oa ho itšoara le maemo
| Karolo | Bolumo |
| BspQ I(10 U/μL) | 1 μL |
| DNA | 1 μg |
| 10 x BspQ I Buffer | 5 μL |
| ke H2O | Ho fihlela ho 50 μL |
Maemo a ho itšoara: 50 ℃, 1~16 h.
Ho se sebetse ha mocheso: 80 ° C bakeng sa metsotso e 20.
Sistimi ea karabelo e khothaletsoang le maemo a ka fana ka phello e batlang e le ntle ea tšilo ea enzyme, e leng sesupo feela, ka kopo sheba liphetho tsa liteko bakeng sa lintlha.
Sesebelisoa sa sehlahisoa
Thibelo endonuclease tshilo ya dijo, Rapid cloning.
Lintlha
1. Molumo oa enzyme ≤ 1/10 ea molumo oa karabelo.
2. Mosebetsi oa linaleli o ka 'na oa etsahala ha mahloriso a glycerol a feta 5%.
3. Ts'ebetso ea ho hlakola e ka etsahala ha Substrate e ka tlase ho tekanyo e khothaletsoang.
