BspQI
Ko te BspQI ka taea te whakaatu ano i roto i te E. coli e mohio ana ki nga waahi motuhake ka mahia i raro
Ko te BspQI, he IIs restriction endonuclease restriction restriction endonuclease, i ahu mai i te recombinant E. coli riaka e mau ana i te ira BspQI kua whakahiato me te whakarereke mai i te Bacillus sphaericus.Ka taea e ia te mohio ki nga waahi motuhake, a ko nga raupapa tohu me nga waahi wehewehe e whai ake nei:
5' · · · ·GCTCTTC(N) · · · · · · · · · · · · · ·3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Nga waahanga hua
1. Mahi teitei, Te nakunaku tere;
2. He iti nga mahi whetu, me te whakarite i te tapahi tika penei i te "scalpel";
3. Kore he BSA me te kararehe-taketake kore utu;
Te Maamaa Methylation
Dahau methylation:Ehara i te mea tairongo;
Dcm methylation:Ehara i te mea tairongo;
CpG Methylation:Ehara i te mea tairongo;
Nga tikanga rokiroki
Me tukuna te hua ≤ 0 ℃;Rokiroki i te -25~- 15 ℃ huru.
Pupuri putunga
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 me 50% glycerol (pH 7.0 @ 25°C).
Whakamaramatanga Waeine
Kotahi te waeine kua tautuhia ko te nui o te whākōkī e hiahiatia ana hei keri i te 1µg o te DNA Mana-roto i roto i te 1 haora i te 50°C i roto i te rahinga tauhohenga katoa o te 50 µL.
Mana Kounga
Whakamātautau Purity Purity (SDS-PAGE):Ko te ma o BspQI ko ≥95% i whakatauhia e te tātaritanga SDS-PAGE.
RNase:Ko te 10U o te BspQI me te 1.6μg MS2 RNA mo te 4 haora i te 50 ℃ karekau he whakahekenga e whakatauhia ana e te electrophoresis gel agarose.
Ngohe DNase Tauwhāiti:Ko te 10U o te BspQI me te 1μg λ DNA i te 50 ℃ mo te 16 haora, ka whakaritea ki te 50 ℃ mo te 1haora, karekau he nui o te DNA i whakatauhia e te electrophoresis gel agarose.
Te Whakakotahi me te Tapahi:Whai muri i te nakunakutanga o te 1 μg λDNA me te 10U BspQI, ka taea te hono nga whatiwhatinga DNA ki te T4 DNA ligase i te 16ºC.A ko enei kongakonga hono ka taea te tapahi ki te BspQI.
E. coli DNA: E. coli 16s rDNA-motuhake TaqMan qPCR kitenga i whakaatu ko E.coli te toenga ira ≤ 0.1pg/ul.
Te toenga pūmua kaihautū:≤ 50 ppm
huakita Endotoxin: LAL-whakamatautau, e ai ki te Hainamana Pharmacopoeia IV 2020 whakaputanga, tikanga whakamatautau tepe gel, ture whanui (1143).Ko te ihirangi endotoxin huakita kia ≤10 EU/mg.
Pūnaha tauhohenga me nga tikanga
| Waehanga | Rōrahi |
| BspQ I(10 U/μL) | 1 μL |
| DNA | 1 μg |
| 10 x BspQ I Kaipupuri | 5 μL |
| dd H2O | Tae atu ki te 50 μL |
Nga tikanga tauhohenga: 50℃, 1~ 16 h.
Te werawera: 80°C mo te 20 min.
Ko te punaha tauhohenga me nga tikanga e taunaki ana ka taea te whakaputa i te huanga nakunaku whākōkī, he tohutoro anake, tirohia nga hua whakamatautau mo nga taipitopito.
Tono hua
Te aukati i te nakunakutanga endonuclease, te tere tere.
Notes
1. Te rōrahi o te whākōkī ≤ 1/10 o te rōrahi tauhohenga.
2. Ka puta pea te mahi whetu ina neke ake i te 5%.
3. Ka puta pea te mahi wehewehe ina te Raarangi kei raro i te ōwehenga e taunakitia ana.
