2×PCR Master Mix (ngaphandle kweDayi)
I-PCR Master Mix iwuhlobo lwekhambi elivamile le-PCR elixutshwe ngaphambili elilungele ukusetshenziswa, okuhlanganisa i-Taq DNA Polymerase, i-dNTP mix MgCl2 kanye nesigcinalwazi esithuthukisiwe.Ngesikhathi sokuphendula, kuphela i-primer nesifanekiso esingangezwa ukuze kukhuliswe, okwenza kube lula kakhulu izinyathelo zokusebenza zokuhlola.Lo mkhiqizo uqukethe ama-stabilizer amahle kakhulu futhi ungagcinwa izinyanga ezi-3 ku-4℃.Umkhiqizo we-PCR une-3'-dA protrusion futhi ungahlanganiswa kalula ube yi-T vector.
Izimo Zokugcina
Umkhiqizo kufanele ugcinwe ku -25 ℃ ~ -15 ℃ iminyaka emibili.
Imininingwane
| Ukwethembeka (vs.Taq) | 1× |
| Isiqalo Esishisayo | No |
| I-Overhang | 3'-A |
| I-Polymerase | I-Taq DNA Polymerase |
| Ifomethi yokusabela | I-SuperMix noma i-Master Mix |
| Isivinini Sokusabela | Okujwayelekile |
| Uhlobo Lomkhiqizo | I-PCR Master Mix (2×) |
Iziyalezo
1.Isistimu yokusabela
| Izingxenye | Usayizi (μL) |
| I-DNA yesifanekiso | Kuyafaneleka |
| I-Primer 1 (10 μmol/L) | 2 |
| I-Primer 2 (10 μmol/L) | 2 |
| I-PCR Master Mix | 25 |
| ddH2O | Kuze ku-50 |
2.I-Amplification Protocol
| Izinyathelo zomjikelezo | Izinga lokushisa (°C) | Isikhathi | Imijikelezo |
| I-pre-denaturation | 94 ℃ | 5 imiz | 1 |
| I-Denaturation | 94 ℃ | 30 isekhondi | 35 |
| Anealing | 50-60 ℃ | 30 isekhondi | |
| Isandiso | 72 ℃ | 30-60sec/kb | |
| Isandiso Sokugcina | 72 ℃ | 10 imiz | 1 |
Amanothi:
1) Ukusetshenziswa kwesifanekiso: 50-200 ng genomic DNA;0.1- 10 ng i-plasmid DNA.
2) uMg2+ukugxila: Lo mkhiqizo uqukethe u-3 mM we-MgCl2 ofanele ukusabela okuningi kwe-PCR.
3) Izinga lokushisa le-annealing: Sicela ubheke inani letiyetha le-Tm lama-Primers.Izinga lokushisa le-anneal lingasethwa libe ngu-2-5 ℃ ngaphansi kwevelu yethiyori ye-primer.
4) Isikhathi sokunwetshwa: Ngokuhlonza i-molecular, 30 sec/kb kuyanconywa.Okwe-gene cloning, 60isekhondi/kb iyanconywa.
Amanothi
1.Ukuze uphephe kanye nempilo yakho, sicela ugqoke amajazi elebhu namagilavu alahlwayo ukuze usebenze.
2.Okokusetshenziswa kocwaningo kuphela!
