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I am trying to design a PCR primer for a gene whose sequence is not known. Even the whole transcriptome sequencing done in our lab did not identify that particular gene. Hence, I guess I am left with only one option: to design the degenerate primers for this gene by performing sequence alignment of the given protein from several related species and designing primers based on conserved region. Could you please explain the process of primer design by this method. I know some steps which I have described below stepwise:

step 1: download sequence of protein in question from related species from NCBI in FASTA format

step 2: perform alignment using clustal omega

step 3: identify the conserved domains

I am not sure how to move forward from here. I tried J-codehop for next steps but it is asking many parameters for which I have no clue. Thanks

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Try going through this tutorial: http://www.scs.illinois.edu/schulten/tutorials/ef-tu/eftu_tutorial.pdf It is a nice introduction on how to use multiseq and identify conserved domains from AA or nt sequences across many taxa.

Also check out http://fungene.cme.msu.edu/ The RDP staff at Michigan State University are the masters of doing exactly what you're trying to do.

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