If you are setting up a (like TaqMan), you need a probe between the primers. Set PRIMER_PICK_INTERNAL_OLIGO=1 . You can set specific constraints for the probe using PRIMER_INTERNAL_... tags (e.g., PRIMER_INTERNAL_OPT_TM ).
# Ubuntu/Debian sudo apt-get install primer3 primer3
Primer3 is a powerful tool for designing primers for PCR applications. Its flexibility, accuracy, and ease of use make it a widely used tool in molecular biology research. By understanding how Primer3 works and how to use it effectively, researchers can design optimal primers for their specific applications. If you are setting up a (like TaqMan),
SEQUENCE_ID=Test_Gene_01 SEQUENCE_TEMPLATE=AGCTGATCGATCGTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTGATCGATCGATCG SEQUENCE_TARGET=20,50 PRIMER_TASK=generic PRIMER_PICK_LEFT_PRIMER=1 PRIMER_PICK_INTERNAL_OLIGO=0 PRIMER_PICK_RIGHT_PRIMER=1 PRIMER_OPT_SIZE=20 PRIMER_MIN_SIZE=18 PRIMER_MAX_SIZE=25 PRIMER_OPT_TM=60.0 PRIMER_MIN_TM=57.0 PRIMER_MAX_TM=63.0 = primer3