The University of Illinois Sequencing Facility offers free primer design to its customers if the primers are subsequently synthesized through the Oligonucleotide Facility. Please contact Laura Guest at lguest@illinois.edu or 217-333-9520 for more information and pricing.

If you would like to design your own custom primer, we recommend that you follow these general recommendations:

1. Primers should be at least 18-20 nucleotides in length to minimize the chances of encountering problems with a secondary hybridization site on the vector or insert.
2. Primers with long runs of a single base should generally be avoided. It is especially important to avoid 3 or more G's or C's in a row.
3. For cycle sequencing, primers with melting temperatures above 50°C generally produce better results than primers with lower melting temperatures.
4. Primers should have a G/C content between 40 and 60 percent. For primers with a G/C content of less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the melting temperature above the recommended lower limit of 50°C.
5. Primers should not contain complementary (palindromes) within themselves; that is, they should not form hairpins. If this state exists, a primer will fold back on itself and result in an unproductive priming event which decreases the overall signal obtained.
6. Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to the other primer used in a PCR reactions (primer dimer formation).
7. If possible, run a computer search against the vector and insert DNA sequences to verify that the primer and especially the 8-10 bases of its 3' end are unique.