Robustart Taq DNA Polymerase

Robustart Taq DNA Polymerase waa billow kulul DNA polymerase.Alaabtani ma aha oo kaliya in ay si fiican u hor istaagto falcelinta aan khaaska ahayn ee ay keento ciribtirka aan gaarka ahayn ee furayaasha ama isugaynta asaasiga ah ee habka diyaarinta iyo kordhinta nidaamka PCR.Sidaa darteed, waxa ay leedahay gaar gaar ah oo aad waxtar u leh kordhinta hab-fiirsiga hoose, waxayna ku habboon tahay falcelinta-weynaynta PCR-ga.Intaa waxaa dheer, badeecadani waxay leedahay dabaqitaan aad u wanaagsan, natiijooyinka kor u qaadista degganna waxaa lagu heli karaa noocyo kala duwan oo falcelin PCR ah.

Qaybaha

1.5 U/μL Robustart Taq DNA polymerase

2.10 × PCR Buffer II (Mg²+ bilaash ah) (ikhtiyaar)

3.25 mm MgCl₂(ikhtiyaar)

* 10 × PCR Buffer II (Mg²+ bilaash ah) kuma jiraan dNTP iyo Mg²+, fadlan ku dar dNTPs iyo MgCl₂marka la diyaarinayo habka falcelinta.

Codsiyada lagu taliyay

1.Kordhinta degdega ah.

2.Kordhinta badan

3.Kordhinta tooska ah ee dhiigga, suufka, iyo shaybaarrada kale.

4.Ogaanshaha cudurrada neef-mareenka.

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.

*Haddii roobabku dhacaan qaboojiyaha ka dib, waa caadi;waxaa lagu talinayaa in la is waafajiyo heerkulka qolka ka hor inta aan la qasin oo la isticmaalin.

Qeexitaan Unug

Hal unug firfircoon (U) ayaa lagu qeexaa sida qadarka ensaymka ee ku dara 10 nmol of deoxyribonucleotide galay walxo aashito- milmi kara at 74°C muddo 30mins ah iyadoo la isticmaalayo shahwada salmon firfircoon DNA sida template/primer.

Xakamaynta tayada

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

2.Dareenka weynaynta, xakamaynta dufcaddii, xasilloonida.

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

Tilmaamaha

Dejinta falcelinta

Xusuusin:

1) ah.Kaydka kuma jiraan dNTP iyo Mg²+, fadlan ku dar dNTPs iyo MgCl₂marka la diyaarinayo habka falcelinta.

2) b.Haddii loo isticmaalo qPCR/qRT-PCR, baarayaasha fluorescent waa in lagu daraa habka falcelinta.Caadi ahaan, fiirsashada ugu dambeysa ee 0.2 μM waxay siin doontaa natiijooyin wanaagsan;haddii waxqabadka falcelinta uu liito, xooga saarista asaasiga ah waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-1 μM.U-fiirsashada baaritaanka waxaa badanaa lagu hagaajiyay 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.

Nidaamka baaskiil wadida kulaylka

Xusuusin

1.Heerka kordhinta polymerase DNA ee degdega ah waa inuusan ka yarayn 1 kb/10 s.Heerkulka kor u kaca iyo hoos u dhaca, habka xakamaynta heerkulka iyo hufnaanta haynta kulaylka ee qalabyada PCR ee kala duwan aad bay u kala duwan yihiin, sidaas darteed waxaa lagu talinayaa in la wanaajiyo xaaladaha falcelinta ugu fiican ee qalabka PCR ee gaarka ah.

2.Nidaamku aad buu u la qabsan karaa, oo leh gaar gaar ah iyo dareen sare.

3.Ku habboon in loo isticmaalo sida dib-u-heleyaasha ogaanshaha PCR ee dareenka sarreeya, waxaana loo isticmaali karaa falcelinta kor u qaadida PCR-ga badan.

4.5′→3′ dhaqdhaqaaqa polymerase, 5′→3′ dhaqdhaqaaqa exonuclease;maya 3′→5′ dhaqdhaqaaqa exonuclease;ma laha shaqo saxid.

5.Ku habboon baaritaanka tayada iyo tirada PCR iyo RT-PCR.

6.Dhamaadka 3′ ee badeecada PCR waa A, kaas oo si toos ah loogu xidhi karo T vector.

7.Habka saddex-tallaabo ayaa lagu talinayaa in loogu talagalay curiyeyaasha heerkulku hooseeyo ama si loo kordhiyo jajabyada ka badan 200 bp.