2. Chemical Components
Also of critical importance is the purity and integrity of template (purified DNA) and primer design. There are several software programs available online for designing primers. The best primers have 18-24 bases, no secondary structure (i.e. hairpin loops), balanced distribution of G/C and A/T pairs, are not complimentary to each other at the 3' ends and have melting temperatures (Tm) about 5-10 degrees celcius below the annealing temperature, which is usually between 55 and 65 degrees celcius. The Tm for both primers should be similar for best results.