2 × PCR Premix （e nang le dae） HYA442
2 × PCR Master Mix e na le Taq DNA Polymerase, dNTPs, le likarolo tse ling tse hlokahalang ke 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 ea setso 'me e loketse ho sebelisoa ka ho eketsa template ea DNA le li-primer. Lihlahisoa tsa PCR li ka kenngoa ka kotloloho bakeng sa electrophoresis ka dae e putsoa ea bromophenol e kentsoeng esale pele. Lihlahisoa tse matlafalitsoeng li na le protrusion ea 3 '-dA 'me li ka kopanngoa habonolo ho T vector. 2 × PCR Master Mix e nolofatsa ts'ebetso ea PCR le ho fokotsa tšilafalo.
Likarolo
| Likarolo | Boholo-1 | Boholo-2 | Boholo-3 |
| 2 × PCR Premix (e nang le dae) | 5mL | 1 mL × 15 | 1 mL × 50 |
Polokelo
Boloka ho -20°C ka lilemo tse 2.
Litaelo
1. Sistimi ea Karabelo
| Likarolo | Molumo |
| 2 × PCR Premix (e nang le dae) | 25 uL |
| Sehlomathiso sa Pele (10 μM) | 2 µL |
| Primer e Khutlisang (10 μM) | 2 µL |
| Template ea DNA* | E feto-fetohang |
| ddH2THE | Ho fihlela ho 50 µL |
* Tšebeliso ea template: 50-200 ng genomic DNA; 0.1-10 ng plasmid DNA
2. Lenaneo la Karabelo
| Mohato | Thempereichara | Nako | Lipotoloho |
| Ho fetoha ha lesoba la pele | 94 ℃ | Metsotso e 5 | 1 |
| Ho fetoha ha letlalo | 94 ℃ | metsotsoana e 30 | 35 |
| Annealing | 50-60 ℃ | metsotsoana e 30 | |
| Katoloso | 72 ℃ | 30-60 metsotsoana/kb | |
| Katoloso ea ho Qetela | 72 ℃ | Metsotso e 10 | 1 |
| Tšoara | 4 ℃ | - | 1 |
1. Mg2+ mahloriso: Sehlahisoa sena se na le 3 mM ea MgCl2, e loketse boholo ba dikarabelo tsa PCR.
2. Mocheso oa ho kenya: Ka kopo sheba boleng ba Tm ba li-primer tse thehiloeng khopolo-taba. Mocheso oa ho kenya o ka beoa ho 2-5℃ ka tlase ho boleng ba khopolo-taba ba primer.
3. Nako ea ho atolosa: Bakeng sa ho tsebahatsa limolek'hule, ho khothaletsoa metsotsoana e 30/kb. Bakeng sa ho etsa hore liphatsa tsa lefutso li tsoe, ho khothaletsoa metsotsoana e 60/kb.
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 hao, ka kopo apara lijase tsa laboratori le liatlana tse lahloang ha u sebetsa.
3. E sebelisetsoa ho etsa lipatlisiso feela!