ExoSAP-IT Express PCR Cleanup Reagents
- Five-minute protocol—fast turnaround time
- One-tube, one-step PCR cleanup— add reagent directly to PCR product
- Novel enzyme technology— irreversibly inactivated in just one minute at 80°C
- Complete sample recovery—100% recovery of PCR products, regardless of amplicon length
- Eliminates spin columns or beads—helps to significantly decrease time and errors while increasing yield
- Compatible—no interference in downstream applications
- Scalable—treat a wide range of volumes
- Multiple formats—single-tube, 8-tube strip, and 96-well plate formats offer flexibility for manual or automated workflows
- Optional tracking dye—helps to avoid losing track of reagent addition during pipetting
Applied Biosystems ExoSAP-IT Express reagents are proprietary mixtures of exonuclease I combined with shrimp alkaline phosphatase (SAP) in a specially formulated buffer, which remove excess primers and dNTPs following PCR. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced during PCR. SAP removes the remaining dNTPs from the PCR mixture that may interfere with subsequent reactions. Adding ExoSAP-IT Express reagent directly to the PCR product eliminates transfer steps to tubes, wells, or columns and conserves amplicons, helping to reduce the chance of cross-contamination; no further processing is needed. After enzymatic cleanup with ExoSAP-IT Express reagent, primers and dNTPs will no longer interfere with DNA sequencing or other downstream applications.
ExoSAP-IT Express reagent offers rapid turnaround times and improved efficiency of resource use while delivering the same superior cleanup as the original ExoSAP-IT reagent. Eliminate spin columns, magnetic beads, sedimentation, filtration, and gel purifications—the novel ExoSAP-IT technology allows for a significant reduction in sample cleanup time with minimal steps, providing the simplest workflow (see figure).
ExoSAP-IT Express reagent vs. other common methods.
A comparison study demonstrated that ExoSAP-IT and ExoSAP-IT Express PCR cleanup reagents offer significant workflow advantages compared to magnetic beads or spin columns while achieving similar results. A novel exonuclease was developed with increased heat sensitivity for PCR amplicon purification in preparation for cycle sequencing. This novel enzyme was the basis for development of the Applied Biosystems ExoSAP-IT Express PCR Product Cleanup Reagent. PCR amplicons of 151, 403 and 634 bp purified enzymatically, by spin column or by magnetic bead separation, all showed similar percent recovery as quantified by fluorescence. Subsequent sequencing using the Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit and capillary electrophoresis (CE) all gave similar results with respect to contiguous read length (CRL) and trace quality. An in-depth sequencing study of only the 634-bp amplicon was carried out with 24 replicate samples purified by ExoSAP-IT Express reagent, ExoSAP-IT reagent, or Agencourt™ AMPure™ XP magnetic beads (Beckman Coulter). Statistical analysis of quality scores showed that out of 4,000 reads, >90% of treated samples had quality values (QVs) above 58 whereas mock-treated samples had >90% of QVs between 30 and 50. By measuring the fluorescence signal beneath each fragment peak, purified samples demonstrated a 4- to 5-fold increase in signal-to-noise ratio. Here we demonstrate that ExoSAP-IT and ExoSAP-IT Express PCR cleanup reagents offer significant workflow advantages compared to magnetic beads or spin columns while achieving similar results.
Treat 5 μl of PCR product with 2 μl of ExoSAP-IT Express reagent. Treatment is carried out at 37°C for 4 minutes followed by an incubation period at 80°C for 1 minute to irreversibly inactivate both enzymes. Once enzyme inactivation is complete, your PCR products are ready for downstream applications such as sequencing (Sanger/NGS), fragment analysis, SNP analysis, in vitro transcription, or single base extension (see figure).
Enzymatic cleanup of PCR reaction with ExoSAP-IT Express reagent.
Which ExoSAP-IT PCR clean-up reagent is right for you?
|ExoSAP-IT Express||ExoSAP-IT reagent (original formulation)||HT ExoSAP-IT reagent|
|Protocol time||5 minutes||30 minutes||14 minutes|
|Throughput level||Low to high; recommended for processing any sample size||Low to mid; recommended for processing 1–96 samples at a time||High; recommended for processing ≥96 samples at a time|
|Platform||Single- or multi-channel pipette, automated liquid handling platforms||Single-channel pipette||Automated liquid handling platforms|
|Stability||–20°C for up to 2 years||–20°C for up to 2 years||–20°C for up to 2 years; once thawed, stable at 4°C for 1 month and at RT for 2 days|
For Research Use Only. Not for use in diagnostic procedures.