2× PCR Master Mix (ntle le Dye)
PCR Master Mix ke mofuta oa tharollo e tloaelehileng ea PCR premixed e seng e loketse ho sebelisoa, ho kenyeletsoa Taq DNA Polymerase, dNTP mix MgCl2 le buffer e ntlafalitsoeng.Nakong ea karabelo, ho ka eketsoa feela primer le template bakeng sa ho holisa, e leng ho nolofatsang mehato ea ts'ebetso ea liteko haholo.Sehlahisoa sena se na le li-stabilizers tse ntle haholo 'me se ka bolokoa likhoeli tse 3 ho 4 ℃.Sehlahisoa sa PCR se na le 3'-dA protrusion mme se ka etsoa habonolo ho T vector.
Maemo a polokelo
Sehlahisoa se lokela ho bolokoa ho -25 ℃ ~ -15 ℃ ka lilemo tse peli.
Litlhaloso
| Botšepehi (vs.Taq) | 1× |
| Hot Qala | No |
| Overhang | 3'-A |
| Polymerase | Taq DNA Polymerase |
| Sebopeho sa Reaction | SuperMix kapa Master Mix |
| Lebelo la ho arabela | Standard |
| Mofuta oa Sehlahisoa | PCR Master Mix (2×) |
Litaelo
1.Reaction System
| Likaroloana | Boholo (μL) |
| DNA ea Setšoantšo | E loketse |
| Primer 1 (10 μmol/L) | 2 |
| Primer 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | Ho ea ho 50 |
2.Amplification Protocol
| Lihato tsa potoloho | Mocheso (°C) | Nako | Lipotoloho |
| Pre-denaturation | 94 ℃ | 5 mets | 1 |
| Denaturation | 94 ℃ | 30 metsotsoana | 35 |
| Annealing | 50-60 ℃ | 30 metsotsoana | |
| Katoloso | 72 ℃ | 30-60sec/kb | |
| Keketso ea ho Qetela | 72 ℃ | 10metsotso | 1 |
Lintlha:
1) Tšebeliso ea template: 50-200 ng genomic DNA;0.1- 10 ng plasmid DNA.
2) Mg2+mahloriso: Sehlahisoa sena se na le 3 mM ea MgCl2 e loketseng liketso tse ngata tsa PCR.
3) Mocheso oa ho futhumatsa: Ka kopo sheba boleng ba theoretical Tm ba Primers.Thempereichara ea annealing e ka beoa ho 2-5 ℃ ka tlase ho boleng ba theory ea primer.
4) Nako ea ho eketsa: Bakeng sa boitsebiso ba molek'hule, 30 sec / kb e buelloa.Bakeng sa gene cloning, 60sec/kb e kgothaletswa.
Lintlha
1.Bakeng sa polokeho le bophelo bo botle ba hau, ka kopo roala lijase tsa lab le liatlana tse lahloang bakeng sa ts'ebetso.
2.Bakeng sa tšebeliso ea lipatlisiso feela!
