Hello seniors! I am stuck in my work now I really need your help. I have a transgenic rice crop. For this, I got positive bands for my transgene in T1 & T2 generation plants. But in Generation 3 I am not getting any single positive band.
What I did do? I did the gradient PCR to check the correct annealing temperature. In my PCR I am getting positive bands for plasmid and for housekeeping gene but no bands for the transgene. Is it Gene's escape? or some other reason? Kindly guide me in this.
Your suggestions will be appreciated.
Dear Aqsa,
It is possible your transgene of interest got silenced in the next generation. Unfortunately it happens and one would have to go back to T2.
However, from your text I wasn't sure if you ran the T1 and T2 PCR products on the same gel as your T3 samples? This would be important to check whether everything technically is the same between the PCRs ran on T1, T2 and T3. I would go back to the original DNA samples and redo the PCR and run the gel at the same time. This would give conclusive evidence it is not a technical artifact, and whether your gene really got silenced. One could also go back to the DNA samples of all generations and try 1 or 2 more primer pairs on your transgene if still unsure.
Good luck!
Sjon
Could be that the plasmids which have lost the gene through deletion have a selective advantage. I would advise doing a PCR with primers across the plasmid insert site to see if the same size band is being produced.