Sanger sequencing: Optimal amount of template and primer

 At the Nevada Genomics Center we offer DNA sequencing using dye-terminator Sanger sequencing with analysis on an Applied Biosystems Prism 3730 DNA Analyzer. A Sanger sequencing reaction is run with a single primer, unlike a polymerase chain reaction (PCR) which is run with two primers. In PCR there is exponential amplification of the amplicon; however in a sequencing reaction, where there is only one primer, there is no exponential amplification. Consequently, the amount of template DNA and primer used to set-up a sequencing reaction should be correct for optimal sequencing.

In considering the amount of template DNA added to a sequencing reaction you must first consider the size, in base pairs, of the template molecule. As the template length increases more DNA is needed to be within the optimal range. The table below lists the recommended amount of DNA template and primer for optimal PCR chemistry and 3730 detection. Note: for plasmid DNA the size is the entire plasmid, vector plus insert. For plasmid DNA you may use the “divide by 20 rule” where you divide the size of the plasmid by 20 to determine the nanograms needed; keeping in mind the maximum is always 1μg. For amplicons you may use the “divide by 50 rule” where you divide the base pair size of the amplicon by 50 to determine the nanograms needed.

Template and primer for plasmid DNA
Template – Plasmid DNA Amount of template
“Divide by 20 rule”
Amount of primer
3000 to 5000bp 150ng to 250ng 2 picomoles = 1ul of 2uM primer
5000 to 10,000bp 250ng to 500ng 10 picomoles = 1ul of 10uM primer
BACs, Cosmids, Fosmids 1μg (maximum) 20 picomoles = 1ul of 20uM primer
Template and primer for PCR amplicons
Template – PCR amplicons Amount of template
“Divide by 50 rule”
Amount of primer
100 to 200bp 4ng 2 picomoles = 1ul of 2uM primer
200 to 500bp 10ng 2 picomoles = 1ul of 2uM primer
500 to 1000bp 20ng 2 picomoles = 1ul of 2uM primer
1000 to 2000bp 40ng 10 picomoles = 1ul of 10uM primer
> 2000bp 50ng 10 picomoles = 1ul of 10uM primer

Important points to remember:

  • Look carefully at the method you use to quantify your template DNA. Is it accurate?
  • Even if you use the optimal amount of template, if the DNA is of poor quality (sheered DNA or contaminated DNA) your sequencing will fail.
  • Using too little OR using too much template DNA can cause the sequencing to fail. The old adage “if one is good than ten must be better” does not hold true when setting up a sequencing reaction.
  • We request a minimum volume of 5uL, so add water if needed to bring the submitted sample up to 5uL. The maximum volume we accept is 25uL, so if your template DNA is too dilute please concentrate it.
  • An important facet to successful sequencing is the ratio of the primer to template molecules. Note in the table that as the nanograms of template increase so does the concentration of primer.
  • Having the correct amount of primer is not going to help if the priming efficiency is poor. Test your primers!