Reagents:

1. Exonuclease I
2. Shrimp Alkaline Phosphatase
3. ddH₂O

The general formula is:

x = (number of PCR reactions)/5
2x = Exonuclease I (uL)
2x = SAP (uL)
6x = ddH₂O (uL)

Mix all three in a tube. Spin down if you have bubbles.
Put 2 ul of mix into 5 uL of PCR product.
Spin down and put in a thermal cycler:

• 37 deg C for 15 minutes
• 80 deg C for 15 minutes
• 4 deg C forever

1. I usually would add 2 or 3 to the number of reactions I had to avoid running out of my dilution as I dispensed into the PCR product.
2. The longer you allow the enzyme to work, the greater the chance that it will start chewing up the ends of your PCR product and consequently, your priming site.
3. This is another website (Washington University School of Medicine Genome Sequencing Center) that has a protocol for diluting Exonuclease I and SAP even more. http://genome.wustl.edu/gsc/Protocols/EXOSAP.shtml