2×PCR Master Mix (aan lahayn dheeh)
PCR Master Mix waa nooc ka mid ah xalka hore ee PCR-ga caadiga ah kaas oo diyaar u ah in la isticmaalo, oo ay ku jiraan Taq DNA Polymerase, dNTP mix MgCl2 iyo kaydka la hagaajiyay.Inta lagu jiro falcelinta, kaliya furaha iyo template ayaa lagu dari karaa si loo kordhiyo, taas oo si weyn u fududaysa tallaabooyinka hawlgalka ee tijaabada.Alaabtani waxay ka kooban tahay xasiliyayaal aad u fiican waxaana lagu kaydin karaa 3 bilood at 4 ℃.Alaabta PCR waxay leedahay soo bixitaan 3′-dA waxaana si fudud loogu xidhi karaa T vector.
Xaaladaha Kaydinta
Alaabta waa in lagu kaydiyaa at -25 ℃ ~ -15 ℃ muddo laba sano ah.
Tilmaamaha
| Fidelity (vs.Taq) | 1× |
| Bilow kulul | No |
| Dulqaadasho | 3′-A |
| Polymerase | Taq DNA Polymerase |
| Qaabka falcelinta | SuperMix ama Master Mix |
| Xawaaraha falcelinta | Heerka |
| Nooca Alaabta | PCR Master Mix (2×) |
Tilmaamaha
1.Nidaamka falcelinta
| Qaybaha | Cabbirka (μL) |
| Qaabka DNA-da | Ku haboon |
| 1-aad (10 μmol/L) | 2 |
| 2-aad (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | Ilaa 50 |
2.Kordhinta Hab-maamuuska
| Tallaabooyinka wareegga | Heerkulka (°C) | Waqtiga | Wareegtada |
| Denaturation ka hor | 94 ℃ | 5 daqiiqo | 1 |
| Denaturation | 94 ℃ | 30 ilbiriqsi | 35 |
| Annealing | 50-60 ℃ | 30 ilbiriqsi | |
| Kordhinta | 72 ℃ | 30-60sek/kb | |
| Kordhinta u dambaysa | 72 ℃ | 10 daqiiqo | 1 |
Xusuusin:
1) Isticmaalka qaabka: 50-200 ng DNA genomic;0.1-10 ng DNA plasmid.
2) Mg2+fiirsashada: Alaabtani waxay ka kooban tahay 3 mM ee MgCl2 oo ku habboon inta badan falcelinta PCR.
3) Heerkulka xajinta: Fadlan tixraac qiimaha Tm ee aragtida aasaasiga ah.Heerkulka xuubka waxaa lagu dejin karaa 2-5 ℃ ka hooseeya qiimaha aragtida asaasiga ah.
4) Waqtiga kordhinta: Aqoonsiga molecular, 30 sek/kb ayaa lagula talinayaa.Marka loo eego hidda-wadaha, 60sec/kb ayaa lagula talinayaa.
Xusuusin
1.Badbaadadaada iyo caafimaadkaaga, fadlan xidho jaakadaha shaybaadhka iyo galoofyada la tuuri karo ee shaqada.
2.Isticmaal cilmi-baaris oo keliya!
