Primer Design Tips

SIZE

• Primers should be at least 18 bases long (20 - 25 preferable) to ensure good hybridization.
• Minimizes the chance of having a secondary hybridization site on the target DNA.
• Necessary for primers with a G-C content less than 50%.
• Greater than 18 base pairs is necessary for A-T rich primers.
• Optimal primer Tm is 55 - 60 ^*C.
• Design primer 10 – 20 bases upstream from site of interest – first 10 - 20 bases do not usually give reliable sequence.

SEQUENCE

• Make sure primer sequence is correct.
• Try to have a GC “clamp” at the 3’ end.
• Avoid runs of greater than 4 bases of an identical nucleotide (especially Gs).
• Keep the G-C content in the range 30-80%, preferably 50-55%.
• PCR Primers: best not to use PCR primers for sequencing – better to use a primer that is more internal.

AVIOD

• Palindromes because they can form secondary structures, which interfere with sequencing.
• Primers that can hybridize to form dimers.
• Secondary structures, especially at the 3’ end.
• Mismatches.
• Hairpin loops within the primer and close to the primer site within the template.
• Potential alternative binding site in the template.

STORAGE

• Store primers dry or in H₂O.
• Make sure to use a fresh dilution of primers each time you sequence.