Applied Biosystems™ ExoSAP-IT Express PCR Product Cleanup with Tracking Dye

Catalog No.	No. of Reactions
A35004	500 Reactions
A35005	1920 reactions

Catalog No. A35004 Supplier Applied Biosystems™ Supplier No. A35004

Description

Our fastest PCR clean-up method - just 5 minutes to superior sequencing results. Now available with optional tracking dye. It is necessary to remove excess primers and unincorporated nucleotides from the PCR reaction prior to sequencing.

• 5 minute protocol: Fastest turnaround time to results
• One-tube, one-step PCR clean-up: Add ExoSAP-IT Express directly to PCR product
• Novel enzyme technology: Irreversibly inactivated in just one minute at 80°C
• Conserve PCR samples: 100% recovery of PCR products, regardless of amplicon length
• Eliminate spin columns or beads: Significantly decreases 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 from manual pipetting to automated workflows
• Go green: Generate less waste with a single-tube solution to PCR clean-up
• Optional tracking dye: Never lose track of reagent addition during pipetting

Specifications

• Content And Storage: Shipped on dry ice. Store at -20°C.
• For Use With (Application): Sequencing, PCR Purification
• No. of Reactions: 500 Reactions
• Product Line: ExoSAP-IT
• Product Type: PCR Product Cleanup
• Protein Form: Recombinant
• Quantity: 500 reactions
• Research Category: Genetic Diseases
• Shipping Condition: Dry Ice

Frequently Asked Questions (FAQs)

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.

Is the tracking dye from ExoSAP-IT Express PCR Product Cleanup with Tracking Dye removed during BigDye Terminator cleanup in Sanger sequencing workflows?

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.

Will the tracking dye in ExoSAP-IT Express PCR Product Cleanup with Tracking Dye affect my downstream applications, like PCR or Sanger sequencing with Big Dye Terminators?

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.

What are the benefits of the tracking dye in ExoSAP-IT Express PCR Product Cleanup with Tracking Dye?

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.

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)

I am looking for the protocol for ExoSAP-IT Express PCR Product Cleanup with Tracking Dye (Cat. No. A35004, A35005). Where can I find it?

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".

Why is it necessary to purify my PCR products prior to the sequencing reaction?

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).

Is the tracking dye removed during BigDye Terminator cleanup in Sanger sequencing workflows?

Yes. Since common techniques for purification after the BigDye Termination reaction involve buffer exchange, the dye is removed during this step.

Does the tracking dye affect downstream processing such as subsequent PCR and Sanger sequencing with BigDye Terminators?

No. The dye has no inhibitory effect on downstream applications.

Does the tracking dye affect the performance or stability of ExoSAP-IT Express reagent?

No. The tracking dye does not affect the performance or stability of ExoSAP-IT Express reagent.

What are the benefits of having a tracking dye?

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.

What is the tracking dye in ExoSAP-IT Express with Tracking Dye?

It is an inert blue color dye for easy tracking of ExoSAP-IT Express reagent as it is pipetted into wells for PCR cleanup.

For Research Use Only. Not for use in diagnostic procedures.