Heerkulka xasaasiga ah UNG

Heerkulka xasaasiga ah UNG (TS-UNG) waa lagu helay muujinta dib-u-habaynta ee E. coli.Insaymku waxa uu kiciyaa sii daynta uracilka bilaashka ah ee DNA-da uracil ka kooban ee hal-iyo laba-geeska ah oo aan firfircoonayn RNA.Marka la barbar dhigo enzyme UNG-ga caadiga ah ee asalka E. coli, enzyme TS-UNG waxay leedahay hawlo sare oo heerkul hooseeya (20 ℃ ~ 37 ℃) waana heerkul xasaasi ah oo si sahlan loo hawlgelin (50℃), isaga oo ka fogaanaya hoos u dhaca xoojinta dUTP-ka kooban Alaabooyinka heerkulka qolka ku jira dhaqdhaqaaqa hadhaaga ah ee sii jiri kara ka dib marka la hawlgeliyo enzyme UNG-ga caadiga ah.Sidaa darteed, enzyme TS-UNG kuma habboona oo kaliya falcelinta ka hortagga faddaraynta PCR, laakiin sidoo kale si fiican ayuu ula jaan qaadayaa barnaamijka kordhinta RT-PCR waxaana loo isticmaali karaa falcelinta ka hortagga faddaraynta RT-PCR.

Codsiga lagu taliyay

Kordhinta Ka-hortagga Wasakhda

Xaalada Kaydinta

-20°C kaydinta muddada dheer, waa in si fiican loo qaso ka hor inta aan la isticmaalin, iska ilaali baraf-dhalaalid joogto ah.

Kaydinta kaydinta

20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, xasiliye, 50% Glycerol.

Qeexitaan Unug

Qadarka enzyme loo baahan yahay si loo dhimo 1µg ee DNA-da hal-xidhaale leh ee ka kooban saldhigyada dU 1 saac 37°C waa 1 unug hawleed (U).

Xakamaynta tayada

1.SDS-PAGE nadiifinta korantada ee ka weyn 98%

2.Dhaqdhaqaaqa hoos u dhaca, xakamaynta dufcaddii, xasiloonida

3.Ma jiro dhaqdhaqaaq nuclease ah oo dibadda ah, ma jiro endonuclease exogenous ama faddarayn exogenous.

Tilmaamaha

Fiiro gaar ah: a: Haddii loo isticmaalo qPCR/qRT-PCR, baaritaanka fluorescent waa in lagu daraa habka falcelinta.Caadi ahaan, fiirsashada ugu dambeysa ee 0.2 μM waxay ku siin kartaa natiijooyin wanaagsan;marka waxqabadka falcelinta uu liito, diirada asaasiga ah waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-1 μM.Caadi ahaan, xoogga saarista baaritaanka waxaa lagu hagaajiyaa inta u dhexeysa 0.1-0.3 μM.Tijaabooyin isugeyn xoog leh ayaa la sameyn karaa si loo helo isku dhafka ugu wanaagsan ee asaasiga ah iyo baaritaanka.

Xusuusin

1.Heerkulka falcelinta ugu fiican ee enzyme TS-UNG waa mid hooseeya, waxaana lagu hagaajin karaa inta u dhexeysa 20 ℃ ~ 37 ℃, qiyaasta enzyme iyo waqtiga falcelinta waxaa lagu hagaajin karaa inta u dhaxaysa 0.1 ~ 0.5 U, 5 ~ 10 daqiiqo;iyo enzymku waa la dami karaa inta lagu gudajiro habka qorista.

2.Waxay ku habboon tahay PCR iyo RT-PCR si looga hortago faddaraynta.

3.Iska ilaali baraf-dhalaalid oo joogto ah, hana u bandhigin isbedbeddelka heerkulka weyn.

4.Hidde-sidayaasha kala duwan ee la xoojiyey waxay leeyihiin waxtarka isticmaalka kala duwan ee dUTP iyo dareenka UNG enzyme, sidaas darteed, haddii isticmaalka nidaamka UNG uu keeno hoos u dhaca dareenka ogaanshaha, nidaamka falcelinta waa in la hagaajiyaa oo la hagaajiyaa, haddii aad u baahan tahay taageero farsamo, fadlan la xiriir. shirkadeena.