I have a fusion protein I am trying to detect. I did RT-PCR and was able to detect the fusion in the primary tissue. When I designed primers on the intron of one of the genes and performed PCR to detect the fusion, I was not able to detect it. Anyone has any suggestions as to why or what can I do to detect the fusion of this chimeric transcript using PCR instead of RT-PCR. Thank you
What's the size of your expected PCR product? And how much genomic DNA did you use as template? May be you have to conduct a long-range PCR using a specific Taq polymerase.
size is about 236 bp and I used 10ng/ul genomic DNA. I am running a lower concentration to see if I can amplify anything. its running right now. I am also trying to PCR the PCR product but it is just weird that I see the band using RT-PCR and I do not see it when I amplify using Regular PCR.
Would it be possible to use the same primers that you used for RT-PCR and then use the size of the product to determine whether it is DNA (longer, with intron) or RNA?
I thought about that. I did do PCR last night on the PCR product that I amplified and did not see anything "PCR ON PCR product" saw a band but very light band. Dont know if that means the chimeric transcript is not expressed as strongly that is why I could not see it with before? Any thoughts?
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