SAP/ExoI treatment of PCR products for sequencing:
Shrimp alkaline phosphatase (SAP) and Exonuclease I treatment can be done on material
directly from a PCR reaction. SAP will work to dephosphorylate any residual primers
and dNTPs (from the PCR) and ExoI will degrade the single stranded molecules 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.
1. After running the PCR sample on a gel (5 µl) to confirm that the reaction worked
and that you have a single product, transfer 8l of the reaction to a new tube.
*NOTE: If the band is very light on the gel you may want to use more of
it in the SAP/ExoI reaction. However, you should make sure to use < 1µg
per SAP reaction so that the efficiency of the enzymes is not
compromised. Be sure to account for the additional template by adjusting
the amount of water used in the master mix described below.
2. Make a Master Mix for the SAP/Exonuclease I
0.05 l Exonuclease I (Epicentre)
0.5 l Shrimp alkaline phosphatase (SAP) – (Roche)
0.5 l SAP 10X buffer
10.3 l sequencing grade (DIUF or 18 ohm) water
Total volume = 11.35 l
1 reaction =
3. Add 10 l of the master mix (above) to the 8 l of PCR product.
4. Spin tubes briefly and put in thermal cycler using the following conditions.
37C – 60 minutes
85C – 15 minutes
4C – HOLD
5. Store samples at 4C until you are ready to sequence the product.
6. For determining the amount needed for sequencing please refer to the instructions
on our website included on the submission form page. We would suggest taking
an absorbance reading (260/280) prior to treatment and estimating the
concentration to use after treatment based on this reading. Alternatively, you may
estimate the concentration of each product using a mass ladder when running the
products on an agarose gel.