I'm currently looking for the efficiency of my primers I have designed.

First, I used 1000, 100, 10, 1, 0.1 ng cDNA as a template. But the result was really awful. It showed very bad both the efficiency and the r-square. (I made it duplicate, and between both Ct results, there was no significant difference. So I assume It was not my pippeting work that bad) 

Then I decide to re-run with the different cDNA template : 4000, 2000, 1000, 500, 250 ng. The result was better, but still bad and also confusing. 

When I tried to find good efficiency by combinating 5 datas that I have, I would get bad r-square value. But when I tried to find good r-square value, I would get bad efficiency.

How could this happen? Can anyone explain? Thank You. 

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