Dear respected colleagues, hi. Im having issues with smearing of my PCR products. I have reduced the DNA, primers, and number of cycles; I have also extracted DNA again using Qiagen kit; However, I cant get any results.
I don't see anything on this gel that looks like a PCR product. Can you tell us more about the experiment? What are your samples, what enzyme or master mix are you using, and what are the cycling conditions? Have these primers been tested before and found to work or are they newly designed?
Hi Mohammad Farzad Afshar, take a look at this link you will find a reason for such PCR pattern: https://www.qiagen.com/be/resources/faq?id=4eb03cc8-4623-4e9e-96b2-6a4c17c03c58&lang=en
Are you running a no template control in your pct? If this control is positive for the smear then you have pcr product contamination in one of your reagents/pipettes and the smear is over amplification of your pcr product
I recommend what Laura Leighton mentioned. It seems that your problem is that no PCR product is there. The smear is not the problem. If you have a positive control sample you can run. Revise your primers, PCR mix, thermal profile, Template and gene of interest. Better to share to solve your problem.
Concentration of primers are not optimal, check the concentration or revise your primers!
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