How do I pass the reference genome to Primer3

Question

[153348734]

I used Primer3 to design the primer, but I don't know how to pass the reference genome file(fasta) to it. This is the help manual : http://primer3.org/manual.html

[Kevin Blighe]

I believe you need to extract the reference sequence yourself, and then paste / import it to Primer3. How to do this? - see here: A: Extract User Defined Region From An Fasta File

[153348734]

Thank you, I'll try it, I'm just starting to learn how to design primers.I had a reference genome and I wanted to take a segment of the sequence and design the primer and consider the specificity of that segment of the primer, and my idea was can I deliver the whole genome to Primer3, so that raised the question.

[i.sudbery]

Primer3 can't handle the entire genome unfortunately, its just too big. You will need to extract the region around where you are trying to design primers (maybe a couple of hundred bp either side of where you want the primers) and design against that. You will then need to separately check whether the designed primers are unique in the genome by searching for other matches (e.g. via blat).

Answer 1

[ATpoint]

You should use PrimerBLAST rather than Primer3 itself. PB uses P3 internally and performs off-target scanning via BLAST against a specified genome.