Shrimp alkaline phosphatase (SAP) and ExonucleaseI treatment is done on the PCR
products to dephosphorylate and degrade any residual primer (from the PCR) so that
the DNA can be used directly for sequencing with your specific sequencing primer.
We have tested whether or not any additional clean up is necessary before
sequencing (i.e. ethanol precipitation) and the data quality was not significantly
improved.
- After running the PCR sample on a gel to confirm that the reaction worked,
transfer 8 µl of this reaction to a new tube.
- Make a master mix for the SAP/Exonuclease I (recipe for 1 reaction below)
- 0.05 µl Exonuclease I (Epicentre)
- 0.5 µl Shrimp alkaline phosphatase (SAP) - (Roche)
- 0.5 µl SAP 10X buffer
- 10.3 µl sequencing grade (DIUF or 18 ohm) water
- Total volume = 11.35 µl
- Add 10 µl of the master mix (above) to the 8 µl of PCR product.
- Spin tubes briefly and put in thermal cycler using the following conditions.
- 37° C - 60 minutes
- 85° C - 15 minutes
- 4° C - HOLD
- Store samples at 4° C until you are ready to sequence the product.
- For sequencing we use 3 µl in a 5 µl reaction.
