2 × PCR Master Mix (na-enweghị agba)
Ngwakọta PCR Master Mix bụ ụdị ihe eji eme PCR emebere nke dị njikere iji, gụnyere Taq DNA Polymerase, dNTP mix MgCl2 na ihe nchekwa kachasị.N'oge mmeghachi omume ahụ, naanị primer na template nwere ike ịgbakwunye maka mmụba, nke na-eme ka usoro nnwale dị mfe.Ngwaahịa a nwere ezigbo stabilizers ma enwere ike ịchekwa ya maka ọnwa 3 na 4 ℃.Ngwaahịa PCR nwere protrusion 3′-dA na enwere ike itinye ya ngwa ngwa na vector T.
Ọnọdụ Nchekwa
Ekwesịrị ịchekwa ngwaahịa ahụ na -25 ℃ ~ -15 ℃ maka afọ abụọ.
Nkọwapụta
| Ikwesị ntụkwasị obi (vs.Taq) | 1× |
| Mmalite na-ekpo ọkụ | No |
| N'ofefe | 3'-A |
| Polymerase | DNA Polymerase |
| Ụdị mmeghachi omume | SuperMix ma ọ bụ Master Mix |
| Ọsọ mmeghachi omume | Ọkọlọtọ |
| Ụdị ngwaahịa | Ngwakọta PCR Master Mix (2×) |
Ntuziaka
1.Sistemụ mmeghachi omume
| Ngwa | Nha (μL) |
| DNA template | Kwesịrị ekwesị |
| Nke mbụ 1 (10 μmol/L) | 2 |
| Nke mbụ 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | Ruo 50 |
2.Protocol Amplification
| Usoro okirikiri | Okpomọkụ (°C) | Oge | okirikiri |
| Tupu denaturation | 94 ℃ | 5mins | 1 |
| Denaturation | 94 ℃ | 30 sk | 35 |
| Na-ewe iwe | 50-60 ℃ | 30 sk | |
| Mgbatị | 72 ℃ | 30-60sk/kb | |
| Mgbatị ikpeazụ | 72 ℃ | Nkeji 10 | 1 |
ndetu:
1) Ojiji ihe atụ: 50-200 ng DNA genomic;0.1-10 ng DNA plasmid.
2) mg2+itinye uche: Ngwaahịa a nwere 3 mM nke MgCl2 dabara maka ọtụtụ mmeghachi omume PCR.
3) Ọnọdụ okpomọkụ: Biko rụtụ aka na uru Tm nke Primers.Enwere ike ịtọ okpomoku na-ekpo ọkụ ka ọ bụrụ 2-5 ℃ dị ala karịa uru atụmatụ nke primer.
4) Oge ndọtị: Maka njirimara molecular, 30 sec / kb ka akwadoro.Maka mkpụrụ ndụ ihe nketa, 60sk/kb ka akwadoro.
Ihe ndetu
1.Maka nchekwa na ahụike gị, biko yiri uwe lab na gloves enwere ike iji rụọ ọrụ.
2.Maka nyocha were naanị!
