
BsaI
BsaI, an IIs restriction endonuclease restriction endonuclease, waxaa laga soo qaatay arecombinant E. coli strain kaas oo xanbaarsan hidda-wadaha BsaI ee cloned iyo wax laga beddelay oo ka yimid Bacillus stearothermophilus. Kuwaas oo leh sifooyin isku mid ah sida enzyme hooyo iyo hoos u dhaca dhaqdhaqaaqa xiddigaha.Siday isugu xigto aqoonsiga iyo goobaha kala-goynta waa sidan soo socota:
5'······GGTCTC(N)·············3'
3'······CCAGAG(NNNN)···5'
Qaybaha
BsaI(20U/μL), 10xBsaI Buffer
Kaydinta
Ku kaydi at -25℃~-15℃, shaqaynaysa 1 sano (iska ilaali wareegyada qaboojinta ee soo noqnoqda).
Kaydinta kaydinta
10mM Tris-HCl, 200mM NaCl, 1mM DTT 0.1mM EDTA, 200µg/ml Recombinant Albumin,50% Glycerol.(pH 7.4 @ 25°C).
Tilmaamaha alaabta
Dhaqdhaqaaq sare, dheefshiidka degdega ah;
1. Dhaqdhaqaaqa xiddiga oo hooseeya, hubinta goynta saxda ah sida "scalpel";
2. Bilaash la'aanta BSA iyo xoolaha asalkoodu yahay.
Dareenka Methylation
Methylation-ka biyo-xireenka: Ma aha xasaasi;
Methylation-ka DCm: Wax u dhimay Qaar ka mid ah Isku-darka Isku-darka;
Methylation CpG: Waxaa xannibay Qaar ka mid ah Isku-dhafka Isku-dhafan.
Qeexitaan Unug
Hal mid oo aan la qeexin ayaa lagu qiyaasaa qiyaasta insaymiska loo baahan yahay si loo dheefshiido 1 µg ee kontoroolka gudaha DNA 1 saac 37°C wadarta mugga falcelinta ee 50 µL.
Xakamaynta tayada
Qiimaynta Nadiifinta Barootiinka (SDS-PAGE)Nadiifinta Bsa I waxay ahayd ≥ 95% waxaa go'aamiyay falanqaynta SDS-PAGE iyadoo la adeegsanayo ogaanshaha Coomassie Blue.
RNase:20 U of Bsa I oo leh 1.6 μg MS2 RNA oo loogu talagalay 16hoursat37℃ wax-soosaarnodegradaationas waxaa go'aamiyay agarose gel electrophoresis.
Waxqabadka DNA-ga Aan Gaarka ahayn:20 Uof BsaI oo leh 1 μg PhiX174 DNA 16 saacadood oo ah 37 ℃ ma keeno DNA xad dhaaf ah sida lagu go'aamiyay agarose gel electrophoresis.
Dib u dhigista iyo dib u dhigista:Ka dib dheefshiidka 1 μg
E.coli DNA:2Uof VacciniavirusCapping Enzyme waxaa lagu baara joogitaanka E. coli genomic DNA iyadoo la isticmaalayo TaqManqPCR oo leh qalabyo u gaar ah E. coli 16S rRNA goobta.E. coli genomic DNA-da wasakhaysan waa ≤1 E. coli genome.
Bakteeriyada Endotoxin:Imtixaanka LAL, marka loo eego daabacaadda ChinesePharmacopoeiaIV2020, habka tijaabada xadka jel, qaanuunka guud (1143).Bakteeriyada endotoxin waa inay noqotaa ≤10 EU/mg.
Nidaamka falcelinta iyo xaaladaha
Qayb | Mugga |
BsaI (20 U/μL) | 1μL |
DNA | 1μg |
10 x BsaI Buffer | 5μL |
dd H2O | Ilaa 50 μl |
Dubo at 37 ° C 15-30 daqiiqo.Dhaqdhaqaaq la'aanta kulaylka:80°C 20 daqiiqo
Nidaamka falcelinta lagu taliyay iyo xaaladaha ayaa ku siin kara saameyn dheefshiidka enzyme aad u wanaagsan, taas oo ah tixraac kaliya, fadlan tixraac natiijooyinka tijaabada ah ee faahfaahinta.
Codsiyada
Xannibaadda Enzyme Dheefshiidka Xakamaynta Degdegga ah.
Qoraalada isticmaalka
1.Mugga enzyme ≤ 1 / 10 ee mugga falcelinta.
2. Dhaqdhaqaaqa-star-ka-shaqsiyeedka waxa uu dhacaa marka fiirsashada glycerol ay ka badan tahay 5%.
3. Dhaqdhaqaaqa nadiifinta ayaa dhici karta marka substrate ka hooseeyo saamiga lagu taliyey.