Primer Design Guidelines for PCR Reactions General types of PCR reactions you may perform: - Gene amplification (with introduction of restriction sites for downstream cloning) - Point mutations - Deletions General considerations for primer design: - Primers should be roughly 17-35 bases in length - Tm (primer melting temperature) ~ 55-70°C • most simply calculated as Tm = [4(G+C) + 2(A+T)]°C • or use a web-based calculator (e. g. http://www.idtdna.com/SciTools/SciTools.aspx) - Primers should end (3’) in a G or C, or CG or GC • improves efficiency of priming - Avoid runs of three or more Cs or Gs at the 3'-ends of primers • may result in mis-priming at G or C-rich sequences - Avoid primer self-complementarity (within a single primer such that 2° structures like hairpins form) - Avoid primer pair complementarities (primer dimers will form and be synthesised preferentially to any other product) - Avoid use of 4 or more consecutive Guanine nucleotides in primer (forms a significantly stable “cruciform” of “guanine tetraplex” 2° structure) Specific considerations for primer design: - Remember to maintain plasmid elements required for protein expression: • In-frame fusions • Stop codons (reverse primer) • Shine Dalgarno sequence (forward primer) - Always consider restriction sites available in the MCS (multiple cloning site) - When engineering restriction sites at ends of an amplified PCR product, always include a few additional bases after the restriction site (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.a sp) Reference where more details can be found: http://www.mcb.uct.ac.za/pcroptim.htm

Primer Design Guidelines for PCR Reactions

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