Primer Design With Primer-BLAST I am trying to design primers for sanger sequencing snp detection. In the past I have used Primer-BLAST, but I'm trying to pick up KRAS codon 12 mutations. The problem is that in Primer-BLAST you specify refseq gene ID and so it doesn't seem to design primers outside of the gene locus. This means that my primers will sit on the SNP. Is there a way to tell Primer-BLAST the exact locus I am trying to sequence? Or is there a better way of doing this? Thanks.

**You can manually restrict the design ranges in Primer-BLAST screen.**

!enter image description here

1. First of all you should enter the targeted sequence. In this case, it will be _NC_000012.12_ which corresponds to chromosome 12 that has KRAS gene.
2. You should determine the position of variant that you interested in that chromosome. The _KRAS G12D_ -or _rs121913529_ \- variant is in 25245350. position. You can find the position of specific variant on dbSNP.
3. For Sanger sequencing, the recommended size is 600-1000 bp. So, we should select ranges +/- 300-500 bp apart from the variant position.
4. For the left primer we choose From:25244850 To:25245050; for the right primer we choose From:25245650 To:25245850. And then submit.
5. Here are the primer-sets that you are looking for. :)



!enter image description here