Leonardo Cardoso Alves

The question is still not clear about what you want to do.

• If you want to use the primer pairs you mentioned you really don't have choice to choose.
• If you want to remoove the degeneracy, you can do that but you have to verify if that combination worked and what does it covers.
• Degenerate primers are a mixture and amplify wide range of fragments.
• What you want to do with reduced number of degeneracy ?
• By the way, while using degenerate primers, it is still one primer pair (forward and reverse) and you don't count it as 64 primers.

if you are amplifying unknown samples to find out what is there then you have to use maximum degeneracy to avoid missing some species which is present. If you are looking specifically for only a few species then you can limit your degeneracy to thos esequences found in your species of interest. Degenerate primers are a mixture of all of the individual primers but because of the way primers are made they come as a mixture of oligos in the same tube at the cost of a single primer so the question of limiting degeneracy is often not possible if you have already made a fully degenerate oligo .

Thank you Paul and Abhijeet,

Think I got it now. So, I am ordering "one" primer whose sequence is 5'-CCTSCSCTTANTDATATGC-3' (ITS4ngsUni) but they are mixing equally in the tube all the combinations. So I'll have 64 different oligos inside one tube, but still, one primer.

Abhijeet, my question was confusing maybe because I didn't understand that earlier.