SEQUENCE_ID=TP53_exon7_test SEQUENCE_TEMPLATE=GGACCTGGTCCTCTGACTGCTCTTTTCACCCATCTACAGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGGTCAGTTGCCCTGAGGGGCTGGCTTCCATGAGACTTCAT PRIMER_TASK=pick_pcr_primers SEQUENCE_TARGET=120,1
PRIMER_MAX_POLY_X=4 # Max mononucleotide repeats (e.g., AAAA) PRIMER_GC_CLAMP=1 # Require G or C in last 5 bases of 3' end PRIMER_MAX_SELF_ANY=4.0 PRIMER_MAX_SELF_END=3.0 PRIMER_NUM_RETURN=5 primer3 input
It signals "end of input" to Primer3. Running It (Command Line) primer3_core < my_primers.txt > my_primers_output.txt Debugging Common Input Errors | Error Message | Likely Fix | | :--- | :--- | | Sequence is shorter than product range | Your SEQUENCE_TEMPLATE is too short. Add flanking bases. | | No valid primers found | Your Tm range is too narrow, or SEQUENCE_TARGET is too close to the end of the template. | | No left primer found | Check PRIMER_MAX_POLY_X or PRIMER_MIN_GC . You are being too strict. | Final Takeaway Primer3 is not a mystery. It is a declarative engine . You define the landscape (sequence) and the constraints (Tm, size, target), and it calculates the best path through the DNA. | | No valid primers found | Your
SEQUENCE_INTERNAL_OLIGO_MIN_SIZE=18 SEQUENCE_INTERNAL_OLIGO_OPT_TM=55.0 Save this as my_primers.txt : | Final Takeaway Primer3 is not a mystery