Primer Design

Primers to be used for automated sequencing must have the correct melting temperature. If the primer/DNA match is 100%, Tm should be 55-60^oC (A,T = 2^oC each; G,C = 4^oC each). Primers, generally, must be 20 bases or longer for use in sequencing reactions. If the match is less complete, Tm must be higher. Avoid primers with long runs of a single base (more than three or four), as well as primers that have secondary structure or can form a dimer. Primer dimer analysis and Tm calculation can be performed using Oligo Analyzer 3.0 on the Integrated DNA Technologies web page. Ensure that your primer concentration is correct and use at least 5 pmol/reaction.

Possible primer problems

• Primer concentration should be 15-26 pmol in the submitted 1.5ml eppendorf tube. A lower amount of primer can cause a reaction to fail.
• Poor primer quality will result in poor sequence quality. N-1 species will cause peaks to be out of frame with one another and basecalling software will not be able to assign correct bases.
• A primer with a melting temperature too low for cycling conditions will not anneal to the template. Design primers with Tm's between 55-60^oC for use at the Genomics Technology Core.
• Secondary structure in the primer will cause the primer to self-anneal and not anneal to the template.
• Mismatch between primer and primer annealing site on the 3' end of a primer can cause the sequencing reaction to fail. If a reaction fails with a standard primer, check to be sure the plasmid contains the primer binding site and that the site was not lost due to restriction digest and cloning of an insert.
• Multiple priming sites on a template will cause peaks that overlap one another. Basecalling software will not be able to assign a base.