BspQI
Hiki ke hōʻike hou ʻia ka BspQI ma E. coli hiki ke ʻike i nā wahi kikoʻī a hana ʻia ma lalo
ʻO BspQI, kahi IIs restriction endonuclease restriction endonuclease, ua loaʻa mai kahi ʻano E. coli recombinant e lawe ana i ka gen BspQI cloned a hoʻololi ʻia mai Bacillus sphaericus.Hiki iā ia ke hoʻomaopopo i nā wahi kikoʻī, a penei ka ʻike ʻana a me nā pae hoʻokaʻawale:
5' · · · · GCTCTTC(N) · · · · · · · · · · 3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Nā hiʻohiʻona huahana
1. Hana kiʻekiʻe, ʻAi wikiwiki;
2. Haʻahaʻa hoku hana, e hōʻoia 'ia oki pololei e like me "scalpel";
3. Me ka loaʻa ʻole o ka BSA a me ke kumu holoholona;
Methylation Sensitivity
Dka methylation:ʻAʻole paʻakikī;
Dknm methylation:ʻAʻole paʻakikī;
CpG Methylation:ʻAʻole paʻakikī;
Nā kūlana mālama
Pono e hoʻouna ʻia ka huahana ≤ 0 ℃;E kūʻai ma -25~- 15 ℃ kūlana.
Paʻa mālama
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 a me 50% glycerol (pH 7.0 @ 25°C).
Wehewehe Unite
Hoʻokahi ʻāpana i wehewehe ʻia e like me ka nui o ka enzyme e koi ʻia e hoʻoheheʻe i 1µg o Internal Control DNA i loko o 1 hola ma 50°C i ka nui o ka nui o ka hopena o 50 µL.
Ka Mana Mana
Hoʻomaʻemaʻe Protein (SDS-PAGE):ʻO ka maʻemaʻe o BspQI he ≥95% i hoʻoholo ʻia e ka SDS-PAGE analysis.
RNase:ʻO 10U o BspQI me 1.6μg MS2 RNA no 4 mau hola ma 50 ℃ ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.
Nā Hana DNase Kūikaʻi ʻole:ʻO 10U o BspQI me 1μg λ DNA ma 50 ℃ no 16 mau hola, i hoʻohālikelike ʻia me 50 ℃ no 1 hola, ʻaʻole e hāʻawi i ka DNA nui e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.
Ligation a me ka ʻoki ʻana:Ma hope o ka ʻai ʻana o 1 μg λDNA me 10U BspQI, hiki ke hoʻopili ʻia nā ʻāpana DNA me ka T4 DNA ligase ma 16ºC.A hiki ke ʻoki ʻia kēia mau ʻāpana ligated me BspQI.
E. coli DNA: E. coli 16s rDNA-specific TaqMan qPCR ka ike ana i ka E.coli genome koena ≤ 0.1pg/ul.
Ke koena protein hoʻokipa:≤ 50 ppm
Bakteri Endotoxin: LAL-hōʻike, e like me ka Chinese Pharmacopoeia IV 2020 edition, gel limit test method, general rule (1143).Pono ka maʻiʻo endotoxin bacteria ≤10 EU/mg.
Pūnaehana pane a me nā kūlana
| ʻāpana | Volume |
| BspQ I(10 U/μL) | 1 μL |
| DNA | 1 μg |
| 10 x BspQ I Hoopaa | 5 μL |
| dd H2O | A hiki i 50 μL |
Nā kūlana pane: 50 ℃, 1 ~ 16 hola.
Hoʻopau wela: 80°C no 20 min.
Hiki i ka ʻōnaehana hopena a me nā kūlana i ʻōlelo ʻia ke hāʻawi i ka hopena digestion enzyme maikaʻi, ʻo ia no ka ʻike wale nō, e ʻoluʻolu e nānā i nā hopena hoʻokolohua no nā kikoʻī.
noi huahana
Kaohi ʻana i ka endonuclease digestion, Hoʻopili wikiwiki.
Nā memo
1. ʻO ka nui o ka enzyme ≤ 1/10 o ka nui pane.
2. Hiki ke hana i ka hoku ke oi aku ka glycerol ma mua o 5%.
3. Hiki ke hana i ka hoʻokaʻawale ʻana ke loaʻa ka substrate ma lalo o ka ratio i ʻōlelo ʻia.
