PRIMER_TARGET=start,end
Historically hosted at the Whitehead Institute/MIT, version 0.4.0 is now commonly accessed via mirrors like the University of Tartu mirror cap T sub m parameters for a difficult sequence? 4.7. Validation of RNA-Seq Data by qRT-PCR - Bio-protocol primer3 input -version 0.4.0-
For SNP genotyping or allele-specific PCR, you may need an internal probe or forced inclusion of a specific base. PRIMER_SEQUENCE_ID=E
PRIMER_SEQUENCE_ID=E.coli_16S_region SEQUENCE=GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGG PRIMER_TASK=pick_detection_primers GC Content : 40–60%
: Allows for "Target" and "Included Region" marking to force primers to flank specific SNPs or exons Secondary Structure Filtering : Built-in checks to avoid primer-dimers or hairpins that can ruin a PCR reaction. Typical Workflow Sequence Input : Paste your DNA sequence in FASTA format or raw text. Define Constraints : Standard guidelines often suggest: : 18–24 bases. GC Content : 40–60%. cap T sub m : 50–65°C (ideally within 5°C of each other). Pick Primers