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View additional product information for ExoSAP-IT™ Express PCR Product Cleanup - FAQs (75001.1.ML, 75001.1.EA, 75001.40.UL, 75001.4X.1.ML, 75001.200.UL, 75001.10.ML)
33 product FAQs found
The HT ExoSAP-IT Fast High-Throughput PCR Product Cleanup reagent is designed for automated handlers as it has a lower viscosity than standard ExoSAP-IT PCR Product Cleanup reagent, which contains some glycerol.
The Quant-iT PicoGreen dsDNA Assay Kit is recommended for quantifying PCR products that have been cleaned with ExoSAP-IT reagent. ExoSAP-IT PCR cleanup results in 100% recovery regardless of amplicon length, and if the PCR product was quantified prior to ExoSAP-IT cleanup, additional quantification is not necessary. We do not recommend using a spectrophotometer as degraded products will be included in the quantification.
For use with reverse transcription, ExoSAP-IT does not degrade the DNA-RNA hybrid. However, it will degrade single-stranded cDNA if a 3' -OH end is present.
The tracking dyes used in the DreamTaq Hot Start Green PCR Master Mix have not been shown to interfere with functionality of the ExoSAP-IT cleanup reagents.
The proofreading function of HiFi DNA polymerases can cause partial degradation of PCR products during extended incubation at 80°C. ExoSAP-IT Express reagent is recommended for HiFi PCR cleanup because it only requires 1 minute at 80°C for inactivation.
ExoSAP-IT-treated PCR products can be used in TA cloning, as the exonuclease I in ExoSAP-IT reagent will not degrade the single A-overhang required for TA cloning.
ExoSAP-IT treatment prior to sequencing helps eliminate miscalls, improves quality scores, increases continuous read length (CRL), and decreases background interference. This yields sequencing results with greater confidence, consistency, and accuracy.
All of our ExoSAP-IT reagents have a similar function for PCR product cleanup, but differ in the areas listed below:
ExoSAP-IT Express PCR Product Cleanup
• Protocol time: 5 minutes
• Format: Single tube*, 8-tube strips, 96-well plate*
• Throughput level: Low to high; Recommended for processing any sample size
• Platform: Single- or multi-channel pipette, automated liquid handling platforms
• Freezes at -20°C: No
• Stability: -20°C for up to 2 years
ExoSAP-IT PCR Proudct Cleanup (our original formulation)
• Protocol time: 30 minutes
• Format: Single tube
• Throughput level: Low to mid; Recommended for processing 1-96 samples at a time
• Platform: Single-channel pipette
• Freezes at -20°C: No
• Stability: -20°C for up to 2 years
HT ExoSAP-IT Fast High-Throughput PCR Product Cleanup (for automated liquid handlers)
• Protocol time: 14 minutes
• Format: Single tube, 8-tube strips, 96-well plate
• Throughput level: High; Recommended for processing ≥96 samples at a time
• Platform: Automated liquid handling platforms
• Freezes at -20°C: Yes
• Stability: -20°C for up to 2 years; once thawed, stable at 4°C for 1 month and RT for 2 days
*Optional tracking dye available (Cat. No. 75002)
ExoSAP-IT reagent has been used to cleanup PCR products prior to processing with the Ion Xpress Plus Fragment Library Kit and Illumina Nextera XT DNA Sample Preparation Kit.
No. ExoSAP-IT reagent does not digest dsDNA, which is why it leaves PCR products and primer dimers undigested. To eliminate primer dimer formation, we recommend that you check for optimal primer design and use PCR polymerases with hot start technology or setup reactions on ice. Additionally, HotStart-IT Binding Protein [Cat. No. 71194400UG] can be added to PCR reactions to prevent primer dimers and increase yield and specificity.
Quant-iT PicoGreen dsDNA Assay Kit is recommended for quantifying PCR products that have been cleaned with ExoSAP-IT reagent. This technique enables equal molar pooling of separate PCR reactions for subsequent amplification.
Yes, as long as the cDNA is in a duplex with RNA. RNase H- (minus) reverse transcriptases, such as SuperScript II/III, are necessary when making cDNA to be cleaned with ExoSAP-IT reagent.
1. 100% recovery-Where bead- and column-based techniques can result in low yields and favor products of certain length, ExoSAP-IT PCR cleanup results in 100% recovery regardless of amplicon length.
2. Faster protocol-Where bead and column-based techniques involve multiple steps and are challenged by throughput, ExoSAP-IT PCR cleanup is a fast (as little as 5 minutes), one-step protocol with unlimited scalability.
3. Enables multiple in-line processing steps-ExoSAP-IT reagent is compatible with most downstream processing steps so workflows can proceed through multiple steps within the same reaction tube.
1. Amplicon-based library prep-ExoSAP-IT reagent is used to remove primers and nucleotides between amplification steps in targeted Amplicon sequencing workflows that use tagged primers to add adapters and indices.
2. Enzymatic shearing of PCR products-ExoSAP-IT reagent is used to remove ssDNA that can interfere with the Enzymatic shearing step by Ion Shear Plus reagent within the Ion Xpress Plus Fragment library Kit.
Yes. ExoSAP-IT reagent can be used to remove primers and nucleotides between amplification steps in NGS workflows that use a PCR-based strategy to amplify targets and add adapters/indices. ExoSAP-IT reagent can also be used for post-library amplification cleanup, but additional processing steps might be required when adapter dimers are prevalent. For additional information on using ExoSAP-IT reagent in NGS, please contact Technical Support at techsupport@thermofisher.com.
Yes. Since common techniques for purification after the BigDye Termination reaction involve buffer exchange, the dye is removed during this step.
No. The dye has no inhibitory effect on downstream applications.
No. The tracking dye does not affect the performance or stability of ExoSAP-IT Express reagent.
It enables visual confirmation of aspiration, dispensing, and reaction mix homogeneity. This is especially useful when using a multi-channel pipette so that you do not lose track of your place on a plate again.
It is an inert blue color dye for easy tracking of ExoSAP-IT Express reagent as it is pipetted into wells for PCR cleanup.
ExoSAP-IT Express reagent contains a novel exonuclease I enzyme that has been engineered to enable rapid, irreversible inactivation at 80°C in only 1 minute.
HT ExoSAP-IT Fast reagent and ExoSAP-IT Express reagent are offered in 8-tube strip and 96-well formats, which are conveniently configured for use with multi-channel pipettes and automated liquid handlers. HT ExoSAP-IT Fast reagent is formulated at a lower viscosity that is more conducive for automation and high-throughput labs where the reagent is consumed in a small number of runs. ExoSAP-IT Express reagent is formulated at a higher viscosity to prevent freezing, which is advantageous when re-using the reagent over multiple runs.
The proofreading function of HiFi DNA polymerases can cause partial degradation of PCR products during extended incubation at 80°C. ExoSAP-IT Express reagent, requiring only 1 minute at 80°C for inactivation, is recommended for HiFi PCR cleanup.
For additional information regarding HiFi PCR cleanup with ExoSAP-IT reagent, please contact Technical Support at techsupport@thermofisher.com.
Yes. In cases where significant primers/dNTPs are leftover, ExoSAP-IT cleanup can be optimized by adding an additional 15 minutes to the 37 degrees C incubation step.
Yes. ExoSAP-IT treatment can remove the primers containing the RNA polymerase promoter that will be used in a subsequent transcription reaction, which ensures that transcription will occur from desired PCR products and not from excess primers.
Yes, but with limited efficiency, as ExoSAP-IT treatment generates mono- and dinucleotides as well as inorganic phosphate and pyrophosphate, all of which can decrease the efficiency of the downstream kinase reaction.
ExoSAP-IT reagent is routinely used for cleaning up PCR products for Sanger sequencing. It is also used for PCR cleanup in workflows that involve downstream primer extension, subsequent rounds of PCR, and denaturation followed by probe hybridization. These applications include SNP analysis, HLA typing, amplicon-based NGS, bead-based arrays, and microarrays.
No. ExoSAP-IT PCR product cleanup results in 100% yield, regardless of amplicon length.
100% recovery. Unlike other techniques such as spin columns, magnetic beads, and precipitation, no yield is lost during ExoSAP-IT treatment and the cleaned PCR products can be used directly in downstream applications.
Yes. Treated PCR products may be stored at -20°C until required.
All versions of ExoSAP-IT reagent follow a simple one-pipette step protocol where 2 µl ExoSAP-IT reagent is added to 5 µl PCR product followed by incubation at 37°C for enzyme activation, then 80°C for inactivation. The incubation times differ as follows:
-ExoSAP-IT reagent (original formulation): 15 minutes at 37°C or 15 minutes at 80°C
-HT ExoSAP-IT Fast reagent: 7 minutes at 37°C or 7 minutes at 80°C
-ExoSAP-IT Express reagent: 4 minutes at 37°C or 1 minute at 80° C
ExoSAP-IT reagent should be stored at -20°C in a non-frost-free freezer.
ExoSAP-IT PCR Product Cleanup is a single-tube reagent for enzymatic cleanup of PCR products. It contains exonuclease I for degradation of unused single-stranded primers and shrimp alkaline phosphatase (SAP) for dephosphorylation of unused nucleotides. Both enzymes are irreversibly inactivated in the ExoSAP-IT protocol so that the cleaned PCR product can be used directly in downstream applications.