2 × PCR Super Mix (ndi Dye)
2 × PCR Master Mix ili ndi Taq DNA Polymerase, dNTPs, ndi zigawo zina zofunika za PCR.Master Mix ndi yokhazikika kwa miyezi itatu pa 4 ℃ ndi zokhazikika zathu.Njira yothetsera kusakanizikana imakonzedweratu kwa PCR wamba ndipo ikukonzekera kugwiritsa ntchito powonjezera template ya DNA ndi zoyambira.Zogulitsa za PCR zitha kuyikidwa mwachindunji kwa electrophoresis yokhala ndi utoto wabuluu wa bromophenol wodzaza kale.Zopangidwazo zimakhala ndi 3 '-dA protrusion ndipo zimatha kupangidwa mosavuta kukhala T vector.2×PCR Master Mix imathandizira kachitidwe ka PCR ndikuchepetsa kuipitsidwa.
Zosungirako
Zogulitsa ziyenera kusungidwa pa -25 ℃~-15 ℃ kwa zaka 2.
Zofotokozera
| Kukhulupirika (vs.Taq) | 1 × pa |
| Hot Start | No |
| Overhang | 3'-A |
| Polymerase | Taq DNA Polymerase |
| Rection Format | SuperMix kapena Master Mix |
| Kuthamanga Kwambiri | Standard |
| Mtundu wa Zamalonda | PCR Master Mix (2x) |
Malangizo
1.Reaction System
| Zigawo | Voliyumu(μL) |
| DNA template | Zoyenera |
| Woyambira 1 (10 μmol/L) | 2 |
| Woyambira 2 (10 μmol/L) | 2 |
| 2 × PCR Master Mix | 25 |
| ddH2O | ku 50 |
2.Amplification Protocol
| Masitepe ozungulira | Kutentha (°C) | Nthawi | Zozungulira |
| Denaturation koyamba | 94 | 5 min | 1 |
| Denaturation | 94 | 30 sec | 35 |
| Annealing | 50-60 | 30 sec | |
| Kuwonjezera | 72 | 30-60 sec/kb | |
| Kuwonjezera komaliza | 72 | 10 min | 1 |
Zindikirani:
1) Kugwiritsa ntchito template: 50-200ng genomic DNA;0.1-10ng plasmid DNA.
2) Mg2+ndende: Mankhwalawa ali ndi 3mM a MgCl2, oyenera machitidwe ambiri a PCR.
3) Kutentha kwapang'onopang'ono: Chonde onani mtengo waukadaulo wa Tm wazoyambira.Kutentha kwa annealing kumatha kukhazikitsidwa ku 2-5 ℃ kutsika kuposa mtengo wongoyerekeza wa primer.
4) Nthawi yowonjezera: Pachizindikiritso cha maselo, 30 sec / kb ikulimbikitsidwa.Pakupanga ma gene, 60 sec/kb ndiyofunikira.
Zolemba
1.Zogulitsa za PCR zokhala ndi 2 × PCR Master Mix sizoyenera polyacrylamide gel electrophoresis.
2.Pa chitetezo ndi thanzi lanu, chonde valani malaya a labu ndi magolovesi otayika kuti mugwire ntchito.
3.Amagwiritsidwa ntchito pofufuza POKHA!
