Accurate high quality data from PCR products
ExoSAP-IT® reagent is designed for simple, quick PCR cleanup for downstream applications, such as CE/Sanger sequencing, TA cloning, and SNP analysis. When PCR amplification is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with these methods. ExoSAP-IT reagent removes these contaminants.
The data below compares sequencing results from PCR product prepared by USB ExoSAP-IT PCR Cleanup and a spin-Column method. The sequencing results from ExoSAP-IT shows superior accuracy with no miscalls or frame shift mutations.
Fluorescent sequencing results of a 100 bp pUC18 PCR fragment sequenced with a -20 Fwd primer using fluorescent sequencing reagents. PCR clean-up performed with: (top) ExoSAP-IT reagent; (bottom) a column designed for PCR cleanup. Base miscalls in (bottom) are due to inherently low yields of short PCR products when using columns.
100% yield from PCR product in one step
As illustrated in the schematic below, ExoSAP-IT reagent is added directly to the PCR product and incubated at 37°C for 15 minutes. After PCR treatment, ExoSAP-IT reagent is inactivated simply by heating to 80°C for 15 minutes. ExoSAP-IT PCR Cleanup utilizes two hydrolytic enzymes, Exonuclease I (Exo I) and Shrimp Alkaline Phosphatase (SAP), in a specially formulated buffer. Exo I enzyme degrades residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. SAP enzymes dephosphorylates the remaining dNTPs, and, thus inactivates them in the PCR reaction.
The enzymatic reaction is done in one step in one tube, and thus provides no product lost, regardless of fragment length, as shown in the figure on the right.
Figure on left: Summary of ExoSAP-IT PCR product treatment.
Figure on right: ExoSAP-IT treatment of PCR products with no sample loss. Single-copy targets were amplified from human genomic DNA. HES-1 (125 bp), numb (455 bp), NRAGE (1.55 kb), and numb (4.6 kb) were loaded on a 1.5% agarose gel before (pre) and after (post) ExoSAP-IT treatment. M is the DNA marker lane. Note that a variety of PCR product sizes can be treated with ExoSAP-IT, even a 125 bp fragment, with no sample loss.
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