Can ExoSAP-IT PCR cleanup reagents be used with automatic handling systems?
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.
After purification with ExoSAP-IT PCR cleanup reagents, what is the recommended quantification method?
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.
Can ExoSAP-IT PCR cleanup reagents be used for removal of primers after first-strand cDNA synthesis?
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.
Will the two tracking dyes used for visualization in the DreamTaq Hot Start Green PCR Master Mix interfere with the ExoSAP-IT PCR cleanup reagents?
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.
Can ExoSAP-IT PCR cleanup reagents be used to cleanup PCR products amplified using High Fidelity (HiFi) DNA polymerases?
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.
Can ExoSAP-IT-treated PCR products be used in cloning applications?
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.
How do the ExoSAP-IT PCR cleanup reagents help improve sequencing results?
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.
What are the differences between the ExoSAP-IT PCR cleanup reagents?
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)
What NGS library preparation kits are compatible with ExoSAP-IT reagent?
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.
Can ExoSAP-IT reagent remove adapter dimers?
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.
How can PCR products be quantified after cleaning with ExoSAP-IT reagent?
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.
Can ExoSAP-IT reagent be used to remove primers and nucleotides from reverse transcribed cDNA in RNA-Seq workflows?
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.
What are the advantages of using ExoSAP-IT reagent to replace bead- or column-based cleanup steps in NGS workflows?
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.
What are the primary uses for ExoSAP-IT reagent in NGS workflows?
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.
Can ExoSAP-IT reagent be used for cleanup steps in next-generation sequencing (NGS) workflows?
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.
How does ExoSAP-IT Express reagent inactivate so quickly at 80 degrees C?
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.
Which version of ExoSAP-IT reagent should be used when cleaning many PCR samples at once?
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.
Can ExoSAP-IT reagent be used to cleanup PCR products amplified using High Fidelity (HiFi) DNA polymerases?
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.
Can ExoSAP-IT reagent be used to cleanup PCR products from multiplex reactions?
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.
Can ExoSAP-IT-treated PCR products be used for in vitro transcription?
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.
Can ExoSAP-IT-treated PCR products be used in 5’-end labeling reactions to make probes?
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.
What are the key applications for ExoSAP-IT reagent?
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.
Is there an amplicon length requirement for ExoSAP-IT reagent?
No. ExoSAP-IT PCR product cleanup results in 100% yield, regardless of amplicon length.
What is the percent recovery of PCR products treated with ExoSAP-IT reagent?
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.
Can PCR products be stored after cleanup with ExoSAP-IT reagent?
Yes. Treated PCR products may be stored at -20°C until required.
What are the differences in the protocols between the different versions of ExoSAP-IT reagent?
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
How should ExoSAP-IT reagent be stored?
ExoSAP-IT reagent should be stored at -20°C in a non-frost-free freezer.
What is ExoSAP-IT PCR Product Cleanup reagent?
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.
