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.

The following table lists the recommended amount of DNA template and primer for optimal Sanger sequencing results. 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