Always look for existing primers. Check scientific journals and primer databases, e.g., primerdb, for existing primer sequences that have been used in other experiments and scientifically demonstrated to work. Unfortunately, because primer design and PCR reactions are difficult processes, professional scientists tend to use primers and reaction parameters that have already been published and experimentally validated.
Find a primer design software. One of the oldest and most widely used is Primer3, which is freely available as a webpage tool. Others include oligocalc, OLIGO 7, PrimerQuest or OligoAnalyzer. Preferably, this should have been a tool recommended to you by a senior scientist currently using it, because all primer design software will have technical challenges and require troubleshooting by an experienced molecular biologist. Upload your target gene sequence into the format, e.g., FASTA or text, specified by whichever software is used.
Select the sequence within your chosen target gene, known as the 'template.' Look at the sequence of several chosen regions for secondary structure (regions of sequence that fold and bind) and avoid these where possible. Also avoid regions with more than four consecutive identical bases, i.e., repeats. Finally, ensure that the current GC, or guanine-cytosine, content does not exceed 60 percent.
Run the primer design software. Specify a primer length of 18 to 24 nucleotides. Set parameters such as melting temperature to be 55 to 80 degrees Celsius, GC content to 40 to 60 percent. Place a 'GC clamp' at the 3'end of the primer sequence by manually typing in a string of 'G' or 'C' nucleotides. Add or remove the 'G' or 'C' bases until the melting temperature is acceptable.
Carry out final checks. These checks depend on the original purpose of the PCR for which the primers are being designed. You can check for self-hybridization or primer-dimer formation, hairpin structures or sequence specificity. For example, if the PCR is to detect single base mutations, then you must verify that your primer recognizes only the template being amplified.