Hi all,

I am conducting a real time PCR for my gene of interest. For the housekeeping gene, I could produce a really good standard curve (R square:0.9 with approximately 105% efficiency and single peak for melting curve). However, the result my gene of interest was not good as the amplification efficiency is too high (higher than 200%) with ct values higher than 30. I have read many articles on how to troubleshoot this problem. One article suggested that too high efficiency might be due to the uncleaned template which contains a lot of inhibitor. As I use undiluted template for my gene of interest, do you think that it might caused by the template contains too much inhibitor?

Another issue is about the primer. Do you think that poor primer design can affect the amplification efficiency?

Please feel free to provide your opinion.

Thanks in advance