I couldn't share the photos in the description directly so I told the story of my problematic PCR in the Question named document. Could you please share your opinion with me if you have enough experience in this field? I'd appreciate it.
dear MR Doğan Tunalı
I think your pollution in the extraction of DNA are very high and you have not any MgCl2 used in the process PCR.
How large of a product are you expecting? 10 seconds is a very short extension time. Usual time is 30 seconds or higher (~1 min per kb).
Doğan Tunalı,
Use the formulation 5X phusion plus Rxn Buffer-5 µl, 10 mM dNTP-0.5 µl, Forward Primer-1.5 µl, Reverse Primer-1.5 µl, Phusion DNA Polimerase-0.2 µl, Template- 3 µl, NF water-upto 25 µl. Use PCR condition: Initial denaturation 98 °C- 3 min, Cycle denaturation 98 °C- 30s, annealing (Gradient) 60-72°C-30 s, elongation 72°C-30s and final elongation 72°C-7 min. Run for 33 cycles. I hope you will get better result.
Thank you A K M Zakir Hossain I will try that. Thank you Katie A S Burnette , I wrote in the Word file that the sequence I want to produce is 5.8kbp long and the annealing time is 10 sec while the extension time is 3 min. Thank you Parivash Layeghi , I will consider that.
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