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 SeqStudio Genetic 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 – 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 – 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 |