Hi,
I have obtained putative transgenic Nipponbare plants by 3 successive selection on antibiotic media. But I am getting negative results for PCR whereas, I got amplification in housekeeping gene and positive control. Do I consider these as wild type plants? Even though grown in antibiotic selection, why it shows PCR-negative?
Thank you
If your housekeeping gene (if from the same batch of isolated gDNA) is positive (= should have no PCR inhibitors), and your positive control is positive (= primers have no problem), then probably they are non-transformants (or selection escapees). Do you have a reporter gene (ex. GUS,...) to use?
Yuan-Yeu Yau
Yes, I have GUS reporter gene. I will be using that for my next transformation. For these plants I had used selection marker as hygromycin and selected for 3 times. Do I have to check for hygromycin gene by PCR?
Supriya Kammar
You can check for hygromycin gene by PCR. But, even you get a band, you probably cannot say that this is definitely a transgenic plant. Because some residual Agrobacterium can give you false positive. But, you can try and tell us the results.
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