2× PCR Super Mix (e nang le Dye)
2 × PCR Master Mix e na le Taq DNA Polymerase, dNTPs, le likarolo tse ling tse hlokahalang tsa PCR.Master Mix e tsitsitse likhoeli tse 3 ho 4 ℃ ka li-stabilizer tsa rona tse ikhethileng.Tharollo ea pele ho motsoako e ntlafalitsoe bakeng sa PCR e tloaelehileng 'me e loketse ho sebelisoa ka ho eketsa template ea DNA le li-primers.Lihlahisoa tsa PCR li ka jarisoa ka kotloloho bakeng sa electrophoresis ka dae e putsoa ea bromophenol e kentsoeng pele.Lihlahisoa tse atolositsoeng li na le 3 '-dA protrusion mme li ka etsoa habonolo ho T vector.2×PCR Master Mix e nolofatsa mokhoa oa PCR le ho fokotsa tšilafalo.
Maemo a polokelo
Lihlahisoa li lokela ho bolokoa ho -25 ℃~-15 ℃ ka lilemo tse 2.
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 (2x) |
Litaelo
1.Reaction System
| Likaroloana | Bolumo(μL) |
| DNA ea Setšoantšo | E loketse |
| Primer 1 (10 μmol/L) | 2 |
| Primer 2 (10 μmol/L) | 2 |
| 2 × PCR Master Mix | 25 |
| ddH2O | ho ea ho 50 |
2.Amplification Protocol
| Lihato tsa potoloho | Mocheso (°C) | Nako | Lipotoloho |
| Denaturation ea pele | 94 | 5 mets | 1 |
| Denaturation | 94 | 30 metsotsoana | 35 |
| Annealing | 50-60 | 30 metsotsoana | |
| Katoloso | 72 | 30-60 sec/kb | |
| Katoloso ea ho qetela | 72 | 10 mets | 1 |
Hlokomela:
1) Tšebeliso ea template: 50-200ng genomic DNA;0.1-10ng plasmid DNA.
2) Mg2+mahloriso: Sehlahisoa sena se na le 3mM ea MgCl2, e loketseng liketso tse ngata tsa PCR.
3) Mocheso oa mocheso: Ka kopo sheba boleng ba theoretical Tm ba li-primers.Thempereichara ea annealing e ka behoa ho 2-5 ℃ tlase ho feta 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, 60 sec/kb e khothaletsoa.
Lintlha
1.Lihlahisoa tsa PCR tse nang le 2 × PCR Master Mix ha lia lokela polyacrylamide gel electrophoresis.
2.Bakeng sa polokeho le bophelo bo botle ba hau, ka kopo roala lijase tsa lab le liatlana tse lahloang bakeng sa ts'ebetso.
3.E sebelisoa bakeng sa tšebeliso ea lipatlisiso FEELA!
