How to Design Primers

1. primers should be 17-28 bases in length; 2. base composition should be 50-60% (G C); 3. primers should end (3’) in a G or C, or CG or GC: this prevents “breathing“ of ends and increases efficiency of priming; 4. Tms between 55-80 C are preferred; 5. 3’-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; 6. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided; 7. runs of three or more Cs or Gs at the 3’-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided. Also keep in mind that most oligonucleotide synthesis reactions are only 98% efficient. This means that each time a base is added, only 98% of the oligos will receive the base. This is not often critical with shorter oligos, but as length increases, so does the probability that a primer will be missing a base. This is very important in mutagenesis or cloning reactions. Purification by HPLC or PAGE is recommended in some cases. #Primers #genetics #DNA #rna