gene quantification

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bosero
bosero's picture
gene quantification

i'm working on realtime PCR and each time i carry out PCR the slope of standard curve is not near -3.32 which corresponds to 100% efficiency.advice me on what o do so as i can achieve slope of -3.32

Ivan Delgado
Ivan Delgado's picture
Hi bosero,

Hi bosero,

There are a number of things you can do but first you need to provide some more details about your assay and how you are preparing your standard curve. 

First of all, if the assay is not designed correctly, you will not get a good slope. Also, the dilutions you use to get your standard curve ideally should be ten-fold dilutions and be of high enough concentration for your assay to easily detect your target. Last but not least most assays are never 100% efficient. As long as you are between 90% and 110%  you should  be fine. 

Good luck