Primer Design:

The choice of sequencing primer will have a large influence on the quality generated from a sample.

Applied Biosystems Recommendations are:

• Avoid primers with long runs of a single base (more than 3 or 4), especially G or C.
• Primers should be between 18 and 30 bases in length - for good hybridisation; lowering the chances of secondary priming sites on the vector or insert.
• The T_m should fall within the range 55^oC to 75^oC.
• The GC content should be between 40% - 60%. If the %GC is below 50, the primer may have to be extended beyond 18 bases to keep the T_m within the recommended range.
• They shouldn't be able to form dimers, or possess secondary structures. They should have low specific binding at the 3' end in order to prevent binding to other sites.
• When designing primers from existing sequence, make sure that the data is good. Remember that the sequence usually starts to get dodgy beyond 450 bases. Please see our page on 'How to Interpret Chromatograms'.
• Position the primer so that the sequence you're trying to obtain falls in the most accurate region of the chromatogram - usually after the first 50 bases, and out to around 450.
• As you cannot count on good data less than 50 bases away from the priming site, if you're trying to sequence a PCR product of less than 250 bases; you may want to consider the following options:

Synthesize your primer with a universal primer attached to the 5'end; eg.the -21M13 forward. Naturally, this does make the whole exercise more expensive!

You may want to consider subcloning your PCR product. See our Template Preparation page.The downside to this is that it will lengthen your experimental time....

You should check your design by using one of the Primer-Design programs available.These include:

Whitehead Institute Primer Ver 3, LaserGene, Oligo, MacVector and the GCG programs.