How to Design a Sequencing Primer

The best sequencing primers are unique sequences that anneal only to the intended stretch of template DNA with accuracy at mid-range temperature. They do not form hairpin loops---that is, back fold and anneal to themselves---nor do they anneal to the other primer (form primer dimers).

Instructions

    • 1

      Design the primer based upon a length of sequence at least fifty nucleotides away from the ends of the template DNA that you wish to extend. You want good overlap.

    • 2

      Choose a sequence that does not have long stretches of any one base, especially not long stretches of Gs or Cs.

    • 3

      Choose a length between 18 to 30 nucleotides long with at least 40 to 60 percent GC content. This will give you an optimal melting point (Tm) range of between 45 to 65 degrees Celsius.

    • 4

      Double-check your sequence against sequences in Genbank to make certain you have not chosen an active site such as a common domain that will prime non-specifically to multiple genes.

    • 5

      Make certain that your primer's 3' (three prime) end is not so heavy in Gs and Cs that it binds to multiple sites.

    • 6

      Write both of your primers in the 5' to 3' (five prime to three prime) direction when ordering. This will require that you write the reverse complement of your reverse primer.

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