I have a problem: I need to design 96 different primer pairs to produce 96 different amplicons from extracted mouse genome. Each amplicon should derive from a specific genomic region, and avoid off-targets.
A list of 96 mouse genomic regions (mm10, format: Ch4:18,405,405 – 18,405,805) that I would like to make an amplicon from.
A table with a Fw and Rv primer pairs that will amplify that region. The primer pair should:
- pass the Primer3 test (control for Tm and CG content and simple things like that)
- pass a BLAST test (control that the primer pair is unlikely to give rise to a secondary amplicons from an off-target region)
This tool already exist: Primer-BLAST available at NCBI. But that system doesn’t allow me to input multiple FASTA files at once. That means that I can spend a day clicking on my browser to get the work done.
I am very confident this problem was solved by people smarter than me, but for the sake of me I haven’t found anything online. I have seen scientists suggesting to use Primer-BLAST from a command line, but I didn’t find instructions for it.
HELP! I am decent at Python, R and API.