I did a QPCR, which gave me the CT around 20 and it had a sharp peak around 80 degree according to the melting curve.
Then, I realized I need to extract DNA from it. So, I ran i5 ul of the 25 ul QPCR in 1% agrose gel in 1 x TAE. The amplicon should be ~750 bp. However, I did not see any band. Instead, I saw smear around the expected position. I don't think it was gel's problem since the ladder and another amplicon (from a QPCR with different primers) were fine.
Have meet this situation? What cause this?
Have you checked your primers? If they have a smear it means there are multiple products - > multiple products mean non specific biding of your primers.
i have been faced this kind of problem before. It might be the specific amplification of your desired target was not going well due to several factors. you can refer to the link below to see some information at the point of smeared bands in PCR process:
http://www.bio-rad.com/en-kr/applications-technologies/pcr-troubleshooting#gel3
Hi
I think that there is more than amplicon around your amplicon in size so there is smear near your size you can check the specifity of your primer by in silico PCR
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