Does anyone have a protocol to evaluate transgene copy-number by quantitative-PCR?

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Rajaguru Bohar

I would be interested to view the reply, as I have the same question

Anwaar Ahmad , Shahina Bano Maqbool , Syed Anwar Hashsham and Mariam B. Sticklen , 2005. Determination of cryIAb and cryIAc Copy Number in Transgenic Basmati 370 Rice (Oryza sativa L.) Plants Using Real-time PCR and its Comparison with Southern Blot. Journal of Biological Sciences, 5: 283-288.

Pl refer

Eugénia De Andrade Silva

Yes. You have to clone one copy of your transgene or one fragment into a plasmid (pUC) toghether with one copy of a single copy endogenous gene. Having a plasmid with a 1:1 transgene:endogenous gene ratio you can perform two calibration curves (one for the transgene; one for the endogenous gene). Use the same dilution series for both calibration curves. Run your sample at the same time that the two calibration curves and infer the transgene copy ratio (in comparison to the choosen endogenous gene). You have to perform some repetitions and in each run do at least 10 replicates of your sample in order to have a good estimate of the uncertainty.