ExoSap clean up of PCR products
Exonuclease plus Shrimp Alcaline Phosphatase (ExoSAP) clean up, as any other PCR purification method aims to remove PCR reagents that unincorporated primers and dNTPs from the PCR reaction so that these do not interfere in a downstream cycle sequencing reaction. Exonuclease will degrade unincorporated primers, while Shrimp Alcaline Phosphatase will dephosphorylate unincorporated dNTPs. PCR products can be purified by adding ExoSap working solution to a PCR product in a ration of 4:10. In reality it works just fine if you add 4 μl of the ExoSap working solution to your 15 μl PCR reaction after you took out 2 μl of the PCR reaction to check on a gel (so to 13 μl of PCR reaction), i.e. use your whole PCR reaction.
1. 13 μl PCR product
2. 4 μl ExoSap working solution
Pipette ExoSap working solution onto PCR product. Just dispense the ExoSap working solution on the side of the tube above the PCR product, use the same tip, no need to mix. It will mix by convection in the thermocycler.
1. 37ºC for 30 min (let ExoSap work)
2. 80ºC for 15 min (inactivate ExoSap)
3. 10ºC for 1 min (cool down block)
ExoSap stock solution (for 600 reactions)
60 μl of Shrimp Alkaline Phosphatase (1 u/μl)
40 μl of Exonuclease I (20 u/μl)
150 μl pure glycerol
150 μl ddH2O
ExoSap work solution
combine ExoSap stock solution with ddH2O in 1:5 ratio