2×PCR Super Mix (oo leh Dye)
2× PCR Master Mix ka kooban Taq DNA Polymerase, dNTPs, iyo qaybaha kale ee PCR-loo baahan yahay.Isku-dhafka 'Master Mix' wuxuu xasilan yahay 3 bilood 4℃ oo leh xasiliyayaal la habeeyey.Xalka-isku-dhafka ka-hor waxaa loo habeeyay PCR-ga caadiga ah oo diyaar u ah in la isticmaalo iyadoo lagu darayo template DNA iyo aasaasayaasha.Alaabta PCR waxaa si toos ah loogu shubi karaa electrophoresis oo leh dheeh buluug ah oo bromophenol ah oo horay loo raray.Alaabooyinka la xoojiyey waxay ka kooban yihiin 3 '-dA protrusion' waxaana si fudud loogu xidhi karaa T vector.Isku dhafka 2×PCR Master Mix wuxuu fududeeyaa habka PCR wuxuuna yareeyaa wasakhda.
Xaaladaha Kaydinta
Alaabta waa in lagu kaydiyaa at -25 ℃ ~ -15 ℃ 2 sano.
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 (2x) |
Tilmaamaha
1.Nidaamka falcelinta
| Qaybaha | Mugga(μL) |
| Qaabka DNA-da | Ku haboon |
| 1-aad (10 μmol/L) | 2 |
| Primer 2 (10 μmol/L) | 2 |
| 2× PCR Master Mix | 25 |
| ddH2O | ilaa 50 |
2.Kordhinta Hab-maamuuska
| Tallaabooyinka wareegga | Heerkulka (°C) | Waqtiga | Wareegtada |
| Denaturation bilowga ah | 94 | 5 daqiiqo | 1 |
| Denaturation | 94 | 30 ilbiriqsi | 35 |
| Annealing | 50-60 | 30 ilbiriqsi | |
| Kordhinta | 72 | 30-60 ilbiriqsi/kb | |
| Kordhinta kama dambaysta ah | 72 | 10 daqiiqo | 1 |
Fiiro gaar ah:
1) Isticmaalka qaabka: 50-200ng DNA genomic;0.1-10ng DNA plasmid.
2) Mg2+fiirsashada: Alaabtani waxay ka kooban tahay 3mM ee MgCl2, oo ku habboon inta badan falcelinta PCR.
3) Heerkulka xajinta: Fadlan tixraac qiimaha Tm-ga aragtida ee aasaasayaasha.Heerkulka xuubka waxaa lagu dejin karaa 2-5 ℃ ka hooseeya qiimaha aragtida asaasiga ah.
4) Waqtiga dheeraadka ah: Aqoonsiga molecular, 30 sek/kb ayaa lagula talinayaa.Xiritaanka hidda-wadaha, 60 sek/kb ayaa lagula talinayaa.
Xusuusin
1.Alaabta PCR ee leh 2× PCR Master Mix kuma haboona jel polyacrylamide electrophoresis.
2.Badbaadadaada iyo caafimaadkaaga, fadlan xidho jaakadaha shaybaadhka iyo galoofyada la tuuri karo ee shaqada.
3.Waxa loo istcimaalayaa cilmi baadhis oo KALIYA!
