Primer3 Patched -

# Ubuntu/Debian sudo apt-get install primer3

To get high-quality primers, you must tune the constraints according to your specific experiment (e.g., standard PCR vs. qPCR).

Untergasser A., et al. (2012). Primer3—new capabilities and interfaces . Nucleic Acids Research, 40(15):e115. primer3

If your template contains known Single Nucleotide Polymorphisms (SNPs), you do not want a primer to bind directly over them, as this might prevent amplification in individuals with the minor allele.

The primer design process in Primer3 involves the following steps: # Ubuntu/Debian sudo apt-get install primer3 To get

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 ).

Understanding the logic Primer3 uses to select primers is essential for troubleshooting failed designs. (2012)

PRIMER_LEFT_0_SEQUENCE=AGCTGATCGATCGTAGCTAG PRIMER_LEFT_0=20,21 (Start Position, Length) PRIMER_LEFT_0_TM=60.5 PRIMER_LEFT_0_GC_PERCENT=50.0 PRIMER_RIGHT_0_SEQUENCE=CTAGCTAGCTAGCTAGCTAG PRIMER_RIGHT_0=50,21 (Start Position, Length - note Right primers are on reverse strand) PRIMER_RIGHT_0_TM=60.2 PRIMER_PAIR_0_PRODUCT_SIZE=31 PRIMER_PAIR_0_PENALTY=0.1234