how to design PCR primer

Question

[mm521]

In PCR process, you don't know the sequence of target DNA, how can you design a primer combined the end of it?

[h.mon]

Please read How To Ask Good Questions On Technical And Scientific Forums. I will have a try at interpreting your question:

You don't know the sequence of the putative target region, but you know the flanking sequences. If so, concatenate the flanking regions with a bunch of Ns in the middle, use primer3 to design primers, and put the Ns inside brackets [NNNNN] so primer3 designs the primers around this region.

Answer 1

[Kevin Blighe]

Edit June 6, 2021: the PDF is no longer accessible. I have put the main content of the SOP here: Lowly undergrad has trouble finding good primers for Professor!

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Please also take a look at my own standard operating procedure for designer primers (and/or a probe), which includes the use of Primer3: https://www2.le.ac.uk/centres/cancer/internal-information/procedures/sops/sop646v01.pdf