2 × PCR Master Mix (popanda utoto)
PCR Master Mix ndi mtundu wa njira wamba wa PCR premixed yankho lomwe ndi lokonzeka kugwiritsidwa ntchito, kuphatikiza Taq DNA Polymerase, dNTP mix MgCl2 ndi buffer yokhathamiritsa.Poyankha, choyambira ndi template yokha ndi yomwe ingawonjezedwe kuti ikulitse, zomwe zimathandizira kwambiri masitepe oyeserera.Izi zili ndi zokhazikika bwino ndipo zitha kusungidwa kwa miyezi itatu pa 4 ℃.Ma PCR ali ndi 3'-dA protrusion ndipo amatha kupangidwa mosavuta kukhala T vector.
Zosungirako
Mankhwala ayenera kusungidwa pa -25 ℃ ~ -15 ℃ kwa zaka ziwiri.
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 (2×) |
Malangizo
1.Reaction System
| Zigawo | Kukula (μL) |
| DNA template | Zoyenera |
| Woyambira 1 (10 μmol/L) | 2 |
| Woyamba 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | ku 50 |
2.Amplification Protocol
| Masitepe ozungulira | Kutentha (°C) | Nthawi | Zozungulira |
| Pre-denaturation | 94 ℃ | 5 mins | 1 |
| Denaturation | 94 ℃ | 30 sec | 35 |
| Annealing | 50-60 ℃ | 30 sec | |
| Kuwonjezera | 72 ℃ | 30-60sec/kb | |
| Final Extension | 72 ℃ | 10 mins | 1 |
Ndemanga:
1) Kugwiritsa ntchito template: 50-200 ng genomic DNA;0.1-10 ng plasmid DNA.
2) Mg2+ndende: Mankhwalawa ali ndi 3 mM ya MgCl2 yoyenera machitidwe ambiri a PCR.
3) Kutentha kwapang'onopang'ono: Chonde onani mtengo waukadaulo wa Tm wa Primers.Kutentha kwa annealing kumatha kukhazikitsidwa ku 2-5 ℃ kutsika kuposa mtengo wongoyerekeza wa primer.
4) Nthawi yowonjezera: Pachizindikiritso cha maselo, 30 sec / kb ikulimbikitsidwa.Kwa gene cloning, 60sec/kb ndiyofunikira.
Zolemba
1.Pa chitetezo ndi thanzi lanu, chonde valani malaya a labu ndi magolovesi otayika kuti mugwire ntchito.
2.Kugwiritsa ntchito kafukufuku kokha!
