ExoSap-IT is an enzymatic cocktail routinely used for PCR product cleanup with 100% product recovery. After PCR amplification, unincorporated dNTPs and primers remaining in the aliquot may interfere with common downstream applications such as cloning and sequencing. This protocol automates ExoSap-IT treatment for PCR purification on the Transcriptic platform. The protocol takes in aliquots of post-PCR reactions containing excess nucleotides and primers, and adds ExoSap-IT reagent in the correct ratio to purify reactions at 15 minutes of incubation at 37C followed by heat inactivation.
This protocol allows the user to select aliquot(s) from their inventory. By default, the entire accessible aliquot volume is purified with the addition of a specific ratio of ExoSap-IT (0.4 * aliquot volume in uL). The reaction is thermocycled for 15 minutes at 37C followed by an inactivation step of 15 minutes at 80C. If a clean-up volume is specified, the specified volume will be transferred to a new plate and purified as described above, leaving you with the original aliquot and a product free from dNTPs and primer contamination. ExoSap-IT execution recovers 100% of the input material and the resulting aliquot can immediately be used for any downstream application.
pUC19 was linearized by PCR to yield a 2385bp product. To test the efficiency of the ExoSap-IT reagent, 30mmoles of dNTPS were added to an aliquot of the PCR product, yielding a total sample volume of 10uL. This aliquot was cleaned up using the ExoSap-IT protocol card followed by Sanger sequencing.
Addition of excess dNTPs causes the sequencing reaction to fail (Figure 1a,b). After ExoSap-IT treatment to remove all excess nucleotides, Sanger sequencing of ExoSap-IT treated product yields clean sequencing traces (Figure 1c).
Transcriptic’s automated platform provides a robust and efficient method for removing dNTPs and primer impurities from your PCR sample. Resulting aliquots are ready-to-go for cloning, Sanger sequencing or other downstream applications.