I-Premix ye-PCR engu-2× （enedayi） HYA442
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 sangaphambi kokuxuba silungiselelwe i-PCR evamile futhi silungele ukusetshenziswa ngokungeza ithempulethi ye-DNA nama-primer. Imikhiqizo ye-PCR ingalayishwa ngqo kwi-electrophoresis ngedayi eluhlaza okwesibhakabhaka ye-bromophenol elayishwe kusengaphambili. Imikhiqizo ekhulisiwe iqukethe i-3 '-dA protrusion futhi ingahlanganiswa kalula ku-T vector. I-2 × PCR Master Mix yenza inqubo ye-PCR ibe lula futhi inciphise ukungcola.
Izingxenye
| Izingxenye | Usayizi-1 | Usayizi-2 | Usayizi-3 |
| I-Premix ye-PCR engu-2 × (enedayi) | 5mL | 1 mL × 15 | 1 mL × 50 |
Isitoreji
Gcina ku--20°C iminyaka emibili.
Imiyalelo
1. Uhlelo Lokusabela
| Izingxenye | Ivolumu |
| I-Premix ye-PCR engu-2 × (enedayi) | 25 uL |
| I-Forward Primer (10 μM) | 2 µL |
| I-Reverse Primer (10 μM) | 2 µL |
| Isifanekiso se-DNA* | Okuguquguqukayo |
| i-ddH2I-THE | Kufika ku-50 µL |
*Ukusetshenziswa kwesifanekiso: 50-200 ng genomic DNA; 0.1-10 ng i-plasmid DNA
2. Uhlelo Lokusabela
| Isinyathelo | Izinga lokushisa | Isikhathi | Imijikelezo |
| Ukuguqulwa kokuqala | 94 ℃ | imizuzu emi-5 | 1 |
| Ukuguquka kwesimo | 94 ℃ | Imizuzwana engama-30 | 35 |
| Ukufakwa kwe-Annealing | 50-60 ℃ | Imizuzwana engama-30 | |
| Isandiso | 72 ℃ | 30-60 imizuzwana/kb | |
| Isandiso Sokugcina | 72 ℃ | imizuzu eyi-10 | 1 |
| Bamba | 4 ℃ | - | 1 |
1. I-Mg2+ ukuhlushwa: Lo mkhiqizo uqukethe i-3 mM ye-MgCl2, ifaneleka kakhulu ekuphenduleni kwe-PCR.
2. Izinga lokushisa lokunamathisela: Sicela ubheke inani le-Tm elingokwemfundiso yama-primer. Izinga lokushisa lokunamathisela lingasethwa libe ngu-2-5℃ ngaphansi kunenani elingokwemfundiso lama-primer.
3. Isikhathi sokwandiswa: Ukuze kutholakale ama-molecule, kunconywa amasekhondi angu-30/kb. Ukuze kukhiqizwe izakhi zofuzo, kunconywa amasekhondi angu-60/kb.
Amanothi
1. Imikhiqizo ye-PCR ene-2× PCR Master Mix ayifaneleki i-polyacrylamide gel electrophoresis.
2. Ukuze uphephe futhi uphile kahle, sicela ugqoke amajazi elebhu kanye namagilavu angasetshenziswa uma uhlinzwa.
3. Isetshenziselwa ucwaningo KUPHELA!