2 × Rapid Taq Super Mix
Nambala ya mphaka: HCR2016A
2 × Rapid Taq Super Mix idakhazikitsidwa ndi Taq DNA Polymerase yosinthidwa, ndikuwonjezera chinthu chokulirapo, chowonjezera chokulitsa komanso makina okhathamiritsa a buffer, ndikuchita bwino kwambiri.Kuthamanga kwa ma tempuleti ovuta monga ma genome mkati mwa 3 kb kumafika 1-3 sec/kb, ndipo ma tempuleti osavuta ngati ma plasmids mkati mwa 5 kb amafika 1 sec/kb.Izi zitha kupulumutsa nthawi ya PCR.Nthawi yomweyo, kusakaniza kumakhala ndi dNTP ndi Mg2+, yomwe imatha kukulitsidwa powonjezera zoyambira ndi ma templates, zomwe zimathandiziranso kwambiri masitepe oyeserera.Kuphatikiza apo, kusakaniza kumakhala ndi utoto wowonetsa ma electrophoretic, womwe ungakhale electrophoresis mwachindunji pambuyo pa zomwe zimachitika.Wotetezera mu mankhwalawa amachititsa kusakaniza kukhalabe ndi ntchito yokhazikika pambuyo pozizira mobwerezabwereza ndi kusungunuka.Gulu la 3'-mapeto A lazinthu za PCR zitha kupangidwa mosavuta mu T vector.
Zigawo
2 × Rapid Taq Super Mix
Zosungirako
Zinthu za PCR Master Mix ziyenera kusungidwa pa -25~-15 ℃ kwa zaka ziwiri.
Zofotokozera
| Mafotokozedwe azinthu | Rapid Taq Super Mix |
| Kukhazikika | 2 × pa |
| Hot Start | Yomanga-Mu Hot Start |
| Overhang | 3'-A |
| Liwiro lakuchita | Mwamsanga |
| Kukula (Chogulitsa Chomaliza) | Mpaka 15 kb |
| Zoyenera mayendedwe | Owuma ayezi |
Malangizo
1. Dongosolo Lochitira (50 μL)
| Zigawo | Kukula (μL) |
| DNA yachitsanzo* | zoyenera |
| Choyambira chakutsogolo (10 μmol/L) | 2.5 |
| Choyambira chosinthira (10 μmol/L) | 2.5 |
| 2 × Rapid Taq Super Mix | 25 |
| ddH2O | ku 50 |
2.Amplification Protocol
| Masitepe ozungulira | Kutentha (°C) | Nthawi | Zozungulira |
| Kusasinthika | 94 | 3 min | 1 |
| Denaturation | 94 | 10 sec |
28-35 |
| Annealing | 60 | 20 sec | |
| Kuwonjezera | 72 | 1-10 sec/kb |
Kovomerezeka kugwiritsa ntchito ma templates osiyanasiyana:
| Mtundu wa template | Gawo logwiritsa ntchito (50 μL reaction system) |
| Genomic DNA kapena E. coli madzi | 10-1,000 ng |
| Plasmid kapena viral DNA | 0.5-50 ng |
| cDNA | 1-5 μL (osapitirira 1/10 ya voliyumu yonse ya machitidwe a PCR) |
| Kugwiritsiridwa ntchito kovomerezeka kwa ma tempulo osiyanasiyana | |
Ndemanga:
1.Kugwiritsa ntchito reagent: sungunuka kwathunthu ndikusakaniza musanagwiritse ntchito.
2. Kutentha kwa Annealing: Kutentha kwa annealing ndi mtengo wapadziko lonse wa Tm, ndipo kungathenso kukhazikitsidwa 1-2 ℃ kutsika kuposa mtengo woyamba wa Tm.
3. Liwiro lowonjezera: Khazikitsani 1 sec/kb kwa ma templates ovuta monga genome ndi E. coli mkati mwa 1 kb;ikani 3 sec/kb pazithunzi zovuta monga 1-3 kb genome ndi E. coli;ikani 10 sec/kb pazithunzi zovuta kupitilira 3 kb genome ndi E. coli.Mutha kuyika mtengo kukhala 1 sec/kb pa template yosavuta monga plasmid yosakwana 5 kb, 5 sec/kb ya template yosavuta monga plasmid pakati pa 5 ndi 10 kb, ndi 10 sec/kb pa template yosavuta. monga plasmid wamkulu kuposa 10 kb.
Zolemba
1. Kuti mutetezeke ndi thanzi lanu, chonde valani malaya a labu ndi magolovesi otayika kuti mugwiritse ntchito.
2. Izi ndizogwiritsidwa ntchito pofufuza POKHA!
