---++ Primer Design ---+++ General guidelines 1 The lengths of primers should be 17-25 bases. 1 GC% should be 40-60% 1 Melting temperature (Tm) should be approximately 50% 1 Usually the 3' base should be a G or C. 1 Ensure that primers cannot hybridize to themselves or each other with a stable structure, especially one that forms continuous base pairing at the 3' end of a primer. ---+++ Tools for primer design 1 <b>[[http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx][OligoAnalyzer]] from [[http://www.idt.com][IDT]]</b><br>Web form that calculates melting temperatures as well as self- and cross-hybridization. 1 <b><nop>PrimerSelect from [[http://www.dnastar.com][DNASTAR]]</b><br> Software program for designing primers. ---+++ Primers for cloning or sequence construction 1 If adding a restriction site to the end of the PCR product, remember to add ~6 extra base pairs for the restriction enzyme.
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Topic revision: r1 - 2008-08-30 - JeffreyBarrick