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View additional product information for ExoSAP-IT Express PCR Product Cleanup with Tracking Dye - FAQs (A35005, A35004)
18 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.
Yes, BigDye Terminator cleanup will remove the tracking dye from the ExoSAP-IT PCR product cleanup reaction. Since common techniques for purification after the BigDye Termination reaction involve buffer exchange, the dye is removed during this step.
No. The tracking dye in ExoSAP-IT Express PCR Product Cleanup with Tracking Dye has not been shown to have inhibitory effect on downstream applications.
The tracking dye enables visual confirmation of aspiration, dispensing, and reaction mix homogeneity, which can also help keep track of wells on a plate. This is especially useful when using a multichannel pipette.
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)
Here is the protocol for ExoSAP-IT Express PCR Product Cleanup with Tracking Dye: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/75001b.pdf
The protocol can be found on the product page for ExoSAP-IT Express PCR Product Cleanup Reagent (https://www.thermofisher.com/order/catalog/product/75001.4X.1.ML) under "Manuals & protocols".
PCR products may contain excess dNTPs and primers. Excess dNTPs will alter the optimized dNTP/ddNTP ratio in the sequencing reaction and may alter signal uniformity. The excess primers may have different priming sites, resulting in sequencing noise. If the PCR primers and sequencing primers are the same, the primer concentration will be higher in the sequencing reaction and may impact signal uniformity of the sample. If the PCR reaction is optimized to exhaust the primers and dNTPs, a PCR purification may not be necessary depending on the application. Please view a list of PCR clean-up kits here (http://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/pcr-product-clean-up.html?cid=fURL-WE4114-pcrcleanup).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
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.