2 × PCR Premix （yokhala ndi Utoto） HYA442
2 × PCR Master Mix ili ndi Taq DNA Polymerase, dNTPs, ndi zinthu zina zofunika pa PCR. Master Mix imakhala yokhazikika kwa miyezi itatu pa 4℃ ndi zokhazikika zathu. Yankho losakanikirana limakhala lokonzedwa bwino kuti ligwiritsidwe ntchito ndi PCR yachikhalidwe ndipo ndi lokonzeka kugwiritsidwa ntchito powonjezera template ya DNA ndi ma primer. Zogulitsa za PCR zitha kuyikidwa mwachindunji kuti zigwiritsidwe ntchito ndi utoto wabuluu wa bromophenol womwe wayikidwa kale. Zogulitsa zomwe zawonjezeredwa zimakhala ndi 3 '-dA protrusion ndipo zimatha kupangidwa mosavuta mu T vector. 2 × PCR Master Mix imapangitsa kuti njira ya PCR ikhale yosavuta komanso imachepetsa kuipitsidwa.
Zigawo
| Zigawo | Kukula-1 | Kukula-2 | Kukula-3 |
| 2 × PCR Premix (yokhala ndi Utoto) | 5mL | 1 mL × 15 | 1 mL × 50 |
Malo Osungirako
Sungani pa -20°C kwa zaka ziwiri.
Malangizo
1. Dongosolo Lochitapo Kanthu
| Zigawo | Voliyumu |
| 2 × PCR Premix (yokhala ndi Utoto) | 25 uL |
| Choyambira Patsogolo (10 μM) | 2 µL |
| Choyambira Chosinthira (10 μM) | 2 µL |
| Chifaniziro cha DNA* | Zosinthika |
| ddH2CHONSE | Mpaka 50 µL |
*Kagwiritsidwe kachithunzithunzi: 50-200 ng genomic DNA; 0.1-10 ng plasmid DNA
2. Pulogalamu Yothandizira
| Gawo | Kutentha | Nthawi | Mayendedwe |
| Kuyamba Kuwonongeka | 94 ℃ | Mphindi 5 | 1 |
| Kuchepa kwa madzi | 94 ℃ | Masekondi 30 | 35 |
| Kuphimba | 50-60 ℃ | Masekondi 30 | |
| Kuwonjezera | 72 ℃ | 30-60 sekondi/kb | |
| Kuwonjezera Komaliza | 72 ℃ | Mphindi 10 | 1 |
| Gwirani | 4 ℃ | - | 1 |
1. Mg2+ kuchuluka: Mankhwalawa ali ndi 3 mM ya MgCl2, yoyenera machitidwe ambiri a PCR.
2. Kutentha kwa annealing: Chonde onani mtengo wa Tm wa primers wongoganizira. Kutentha kwa annealing kumatha kuchepetsedwa kufika pa 2-5℃ kuposa mtengo wa annealing wa primers.
3. Nthawi yotalikira: Kuti mudziwe kuchuluka kwa mamolekyu, masekondi 30/kb ndi ofunikira. Kuti mupange ma gene cloning, masekondi 60/kb ndi ofunikira.
Zolemba
1. Zogulitsa za PCR zokhala ndi 2× PCR Master Mix sizoyenera kugwiritsa ntchito polyacrylamide gel electrophoresis.
2. Kuti mukhale otetezeka komanso athanzi, chonde valani majekete a labu ndi magolovesi ogwiritsidwa ntchito nthawi iliyonse mukamachita opaleshoni.
3. Imagwiritsidwa ntchito pofufuza kokha!