How do I design PCR primers and hybridization oligonucleotides?

Program name: eprimer3     

(molbiol username / password required)

Notes: eprimer3 is an interface to the well established program, "primer3".

Troubleshooting: what to do if eprimer3 cannot find any primers? (From the eprimer3 manual)

• Set the "explain flag" option (in the advanced section) to "yes" - eprimer3 will then attempt to explain why no primers were chosen.
• Try relaxing various parameters, including the self-complementarity parameters and max and min oligo melting temperatures. For example, for very A-T-rich regions you might have to increase maximum primer size or decrease minimum melting temperature.
• It is usually unwise to reduce the minimum primer size if your template is complex (e.g. a mammalian genome), since small primers are more likely to be non-specific.
• Make sure that there are adequate stretches of non-Ns in the regions in which you wish to pick primers. If necessary you can also allow an N in your primer and use an oligo mixture containing all four bases at that position.

Further reading: eprimer3 background