Designing PCR primers

1.length of individual primers between 18-24 bases. Longer primers (30-35 bp) seem to work in more similar cycling conditions compared with shorter primers, and can make multiplexing easier;

2.it is desirable (but not absolutely necessary) that the two primers have a close melting temperature or Tm (say, within 5℃ or so). If Tm difference between the two primers is high, the lower Tm can be increased by increasing the length of that primer at the 3‘ end (this can also keep the size of the amplified locus constant) or the 5’ end;

3.purine:pyrimidine content around 1:1 (maybe 40-60%);

4.if possible, primer sequence should start and end with 1-2 GC pairs.