2×PCR Super Mix (enedayi)
I-2× PCR Master Mix iqukethe i-Taq DNA Polymerase, i-dNTPs, nezinye izingxenye ezidingekayo ze-PCR.I-Master Mix izinzile izinyanga ezi-3 ku-4℃ ngeziqinisi zethu ezenziwe ngokwezifiso.Isixazululo se-pre-mix senzelwe i-PCR evamile futhi silungele ukusetshenziswa ngokwengeza ithempulethi ye-DNA neziqalo.Imikhiqizo ye-PCR ingalayishwa ngokuqondile nge-electrophoresis ngodayi oluhlaza we-bromophenol olayishwe ngaphambili.Imikhiqizo ekhulisiwe iqukethe i-3 '-dA protrusion futhi ingahlanganiswa kalula ibe yi-T vector.I-2×PCR Master Mix yenza inqubo ye-PCR ibe lula futhi yehlise ukungcoliswa.
Izimo Zokugcina
Imikhiqizo kufanele igcinwe ku -25℃~-15℃ iminyaka emi-2.
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 (2x) |
Iziyalezo
1.Isistimu yokusabela
| Izingxenye | Ivolumu(μL) |
| I-DNA yesifanekiso | Kuyafaneleka |
| I-Primer 1 (10 μmol/L) | 2 |
| I-Primer 2 (10 μmol/L) | 2 |
| 2× PCR Master Mix | 25 |
| ddH2O | kuye 50 |
2.I-Amplification Protocol
| Izinyathelo zomjikelezo | Izinga lokushisa (°C) | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 94 | 5 imiz | 1 |
| I-Denaturation | 94 | 30 isekhondi | 35 |
| Anealing | 50-60 | 30 isekhondi | |
| Isandiso | 72 | 30-60 isekhondi/kb | |
| Isandiso sokugcina | 72 | 10 imiz | 1 |
Qaphela:
1) Ukusetshenziswa kwesifanekiso: 50-200ng i-genomic DNA;0.1-10ng i-plasmid DNA.
2) uMg2+ukugxila: Lo mkhiqizo uqukethe i-3mM ye-MgCl2, ilungele ukusabela okuningi kwe-PCR.
3) Izinga lokushisa le-annealing: Sicela ubheke inani letiyetha le-Tm lama-primers.Izinga lokushisa le-annealing lingasethwa libe ngu-2-5℃ ngaphansi kwevelu yethiyori ye-primer.
4) Isikhathi sokunwetshwa: Ngokuhlonza i-molecular, 30 sec/kb kuyanconywa.Ngokwenza i-gene cloning, 60 sec/kb kuyanconywa.
Amanothi
1.Imikhiqizo ye-PCR ene-2× PCR Master Mix ayifanele i-polyacrylamide gel electrophoresis.
2.Ukuze uphephe kanye nempilo yakho, sicela ugqoke amajazi elebhu namagilavu alahlwayo ukuze usebenze.
3.Isetshenziselwa ucwaningo KUPHELA!
