2× Ngwa ngwa Taq Super Mix
Mpempe nwamba: HCR2016A
2 × Rapid Taq Super Mix dabere na Taq DNA Polymerase gbanwere, na-agbakwunye ihe ndọtị siri ike, ihe nkwalite nkwalite yana sistemụ nchekwa kachasị, yana arụmọrụ nkwalite dị elu.Ọsọ mmụba nke ndebiri mgbagwoju anya dị ka genome n'ime 3 kb ruru 1-3 sec/kb, na nke ndebiri dị mfe dị ka plasmids n'ime 5 kb ruru 1 sec/kb.Ngwaahịa a nwere ike ịchekwa oge mmeghachi omume PCR nke ukwuu.N'otu oge ahụ, ngwakọta nwere dNTP na Mg2+, nke enwere ike ịbawanye naanị site na ịgbakwunye primers na ndebiri, nke na-emekwa ka usoro ọrụ nke nnwale ahụ dịkwuo mfe.Ọzọkwa, mix nwere electrophoretic egosi agba, nke nwere ike ịbụ kpọmkwem electrophoresis mgbe mmeghachi omume.Onye na-ahụ maka nchekwa dị na ngwaahịa a na-eme ka ngwakọta ahụ nọgide na-arụ ọrụ kwụsiri ike ka ọ kwụsịrị ugboro ugboro na agbaze.Enwere ike ijikọ eriri 3'-ọgwụgwụ A nke ngwaahịa PCR na vector T.
Ngwa
2× Ngwa ngwa Taq Super Mix
Ọnọdụ Nchekwa
Ekwesịrị ịchekwa ngwaahịa PCR Master Mix na -25 ~ -15 ℃ maka afọ 2.
Nkọwapụta
| Nkọwapụta ngwaahịa | Ngwa ngwa Taq Super Mix |
| Ntinye uche | 2× |
| Mmalite na-ekpo ọkụ | Mmalite Hot arụnyere n'ime ya |
| N'ofefe | 3'-A |
| Ọsọ mmeghachi omume | Ngwa ngwa |
| Nha (ngwaahịa ikpeazụ) | Ihe ruru 15kb |
| Ọnọdụ maka njem | Ikpụrụ kpọrọ nkụ |
Ntuziaka
1. Sistemụ mmeghachi omume (50 μL)
| Ngwa | Nha (μL) |
| DNA ndebiri* | dabara adaba |
| Ntugharị mbugharị (10 μmol/L) | 2.5 |
| Ntugharị azụ (10 μmol / L) | 2.5 |
| 2× Ngwa ngwa Taq Super Mix | 25 |
| ddH2O | ruo 50 |
2.Protocol Amplification
| Usoro okirikiri | Okpomọkụ (°C) | Oge | okirikiri |
| Predenaturation | 94 | Nkeji 3 | 1 |
| Denaturation | 94 | 10 sk |
28-35 |
| Na-ewe iwe | 60 | 20 sk | |
| Mgbatị | 72 | 1-10 sk/kb |
Eji ndebiri dị iche iche akwadoro:
| Ụdị template | Oke ojiji nke ngalaba (sistemụ mmeghachi omume 50 μL) |
| Genomic DNA ma ọ bụ E. coli mmiri mmiri | 10-1,000 n'arọ |
| Plasmid ma ọ bụ DNA viral | 0,5-50 n'arọ |
| cDNA | 1-5 µL (ọ bụghị ihe karịrị 1/10 nke mkpokọta olu mmeghachi omume PCR) |
| Eji ndebiri dị iche iche akwadoro | |
ndetu:
1.Ojiji reagent: gbazee nke ọma ma gwakọta tupu ejiri ya.
2. Annealing okpomọkụ: The annealing okpomọkụ bụ eluigwe na ala Tm uru, na nwekwara ike ịtọ 1-2℃ ala karịa primer Tm uru.
3. Ọsọ ndọtị: Tọọ 1 sec / kb maka ndebiri mgbagwoju anya dị ka genome na E. coli n'ime 1 kb;tọọ 3 sk/kb maka ndebiri mgbagwoju anya dị ka 1-3 kb genome na E. coli;tọọ 10 sk/kb maka ndebiri mgbagwoju anya karịrị 3 kb genome na E. coli.Ị nwere ike ịtọ uru na 1 sec / kb maka ndebiri dị mfe dị ka plasmid na-erughị 5 kb, 5 sec / kb maka template dị mfe dị ka plasmid n'etiti 5 na 10 kb, na 10 sec / kb maka ndebiri dị mfe. dị ka plasmid ibu karịa 10 kb.
Ihe ndetu
1. Maka nchekwa gị na ahụike gị, biko yie mkpuchi ụlọ nyocha na gloves enwere ike iji rụọ ọrụ.
2. Ngwaahịa a bụ maka nyocha naanị!
