BspQI
BspQI irashobora kugaragazwa muri E. coli ishobora kumenya imbuga runaka kandi ikorerwa munsi
BspQI, IIs ibuza endonuclease kubuza endonuclease, ikomoka kumurongo wa recombinant E. coli itwara gen ya BspQI yakoronijwe kandi yahinduwe kuva Bacillus sphaericus.Irashobora kumenya imbuga zihariye, hamwe no kumenya urutonde hamwe na site ya clavage nibi bikurikira:
5 '· · · · · GCTCTTC (N) · · · · · · · · · · · · 3'
3 '· · · · · CGAGAAG (NNNN) · · · · 5'
Ibiranga ibicuruzwa
1. Igikorwa kinini, Kurya vuba;
2. Igikorwa cyo hasi yinyenyeri, kwemeza gukata neza nka "scalpel";
3. Hatariho BSA ninkomoko yinyamanswa kubuntu;
Methylation Sensitivity
Dam methylation:Ntabwo Yumva;
Dcm methylation:Ntabwo Yumva;
CpG Methylation:Ntabwo Yumva;
Imiterere yo kubika
Ibicuruzwa bigomba koherezwa ≤ 0 ℃;Ubike kuri -25 ~ - 15 ℃ imiterere.
Ububiko
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).
Igisobanuro
Igice kimwe gisobanurwa nkubunini bwa enzyme isabwa kugirango igogwe 1µg ya ADN igenzura imbere mumasaha 1 kuri 50 ° C mubunini bwa reaction ya 50 µL.
Kugenzura ubuziranenge
Isuzuma rya poroteyine (SDS-PAGE):Ubuziranenge bwa BspQI bwari ≥95% byagenwe nisesengura rya SDS-PAGE.
RNase:10U ya BspQI hamwe na 1.6μg MS2 RNA mu masaha 4 kuri 50 ℃ nta musaruro ugabanuka nkuko byagenwe na agarose gel electrophorei.
Igikorwa kidasanzwe cya DNase:10U ya BspQI hamwe na 1μg λ ADN kuri 50 ℃ mu masaha 16, ugereranije na 50 ℃ kuri 1hour, nta ADN irenze nkuko byagenwe na agarose gel electrophorei.
Kuburana no Gusubiramo:Nyuma yo gusya 1 μg λDNA hamwe na 10U BspQI, ibice bya ADN birashobora guhuzwa na T4 ADN ya ligase kuri 16ºC.Kandi ibi bice byahujwe birashobora gusubirwamo hamwe na BspQI.
E. coli ADN: E. coli 16s rDNA yihariye ya TaqMan qPCR yerekanye ko ibisigisigi bya E.coli ≤ 0.1pg / ul.
Ibisigarira bya poroteyine:≤ 50 ppm
Indwara ya bagiteri Endotoxin: Ikizamini cya LAL, ukurikije Pharmacopoeia yo mu Bushinwa IV 2020, uburyo bwo gupima gel, uburyo rusange (1143).Indwara ya bagiteri igomba kuba ≤10 EU / mg.
Sisitemu yo kwitwara hamwe nibisabwa
| Ibigize | Umubumbe |
| BspQ I (10 U / μL) | 1 μL |
| ADN | 1 μg |
| 10 x BspQ I Buffer | 5 μL |
| dd H2O | Kugera kuri 50 μL |
Imiterere yo kwitwara ： 50 ℃, 1 ~ 16 h.
Gushyushya kudashyira ： 80 ° C kuminota 20.
Sisitemu isabwa ya reaction hamwe nibisabwa birashobora gutanga ingaruka nziza ya enzyme igogorwa, igenewe gusa, nyamuneka reba ibisubizo byubushakashatsi kubisobanuro birambuye.
Gusaba ibicuruzwa
Kubuza endonucisha igogora, cloni yihuse.
Inyandiko
1. Ingano ya enzyme ≤ 1/10 yubunini bwa reaction.
2. Igikorwa cyinyenyeri gishobora kubaho mugihe intumbero ya glycerol irenze 5%.
3. Igikorwa cya clavage gishobora kubaho mugihe Substrate munsi yikigereranyo cyasabwe.
