I work in plant virus and I use a specific primer to make the cDNA (I do not use oligo dT or hexamer) and it works giving me a sharp band (photo in attachment)
But when I made the PCR using these primers it does not work giving me that photo
Note: all samples are the same except for annealing temp.
I really appreciate any help you can provide.
I presume you have a primer pair and not a single primer. How do you know the clones you are screening actually have your cDNA clone? Maybe you aren't getting PCR product because there is no insert.
I assume that I have my clone because a cDNA band appear and the only thing I use for cDNA synthesis is the reverse primer which is specific to my virus (I check it several time in NCBI)
Yasmeen Abuhadema, something about your answer doesn't make sense. Do you have plasmid preps of your presumed clone that you are using as templates to test for the insert?
You need 2 primers for a PCR amplification, you can't amplify with only a single primer.
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