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Services – ExoSAP-IT™ Express


• ExoSAP-IT™ Express reagent

• ExoSAP-IT vs. EtOH-EDTA precipitation

• Online Submission Request

• Physical Submission of Samples

ExoSAP-IT™ Express reagent

Before sequencing PCR products, residual primers and unincorporated nucleotides must be removed from the samples. To accomplish this, this product uses two hydrolytic enzymes: a modified Exonuclease I; and, Shrimp Alkaline Phosphatase [SAP]. For full product details, visit the ThermoFisher website: ExoSAP-IT Express [A35004]).

In brief, ExoSAP-IT is active in commonly used PCR buffers, so no buffer exchange is required and the reaction occurs in a single tube with just one pipetting step. The reagent is added directly to the PCR product for a 4 minute cleanup at 37oC, followed by a 1 minute inactivation at 80oC; an inert tracking dye offers visual confirmation that the reagent has been added. Finally, none of the kit components will interfere with downstream applications.

ExoSAP-IT vs. EtOH-EDTA precipitation

There are pros-&-cons to using ExoSAP-IT Express vs. the Core's 'primer-depletion' EtOH-EDTA precipitation protocols.

  • Pro: This kit retains 100% of the original PCR product... whereas one can expect to lose some (usually <20-30%) of the PCR products in the EtOH-EDTA procedures.
  • Pro: Because ExoSAP-IT retains 100% of dsDNA products, it can be used as a troubleshooting tool when there are concerns that the EtOH-EDTA protocols are losing too much PCR product.
  • Con: Unlike the EtOH-EDTA primer-depletion protocols, ExoSAP-IT will not remove primer-dimers or other non-specific small PCR products. If present in the sample, such small non-targets will create noise in the early sequence data.
  • Con: Unless doing just a few samples, it is much more expensive to use this kit.

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    Online Submission Request

  • Log In to the website, and click on "Services Page":
    • Make the necessary 'selections'.
    • In the "Client Memo" field, state the volume of PCR product (or appropriately diluted PCR product) that was provided (see comments under 'Physical Submission') and include any other information deemed pertinent to processing your samples.
    • Click the 'check box', and submit your request.
  • If you make an error in your request, you may revise that request if you do so before we begin processing it.
  • You will receive an automated email when your samples are processed and ready for pick-up.

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    Physical Submission of Samples

  • Tube or Plates?:  Samples may be provided in either 8-strip PCR tubes or in 96-well PCR plates; both tubes and plates need to be the standard size (i.e., 0.2-ml volume). Do not submit samples in individual tubes or in sizes other than 0.2-ml.
  • Sample mass:  To achieve good sequencing results, the template input needs to fall in the right mass range. As a guide, for a 500-bp fragment, ˜2-6 ng is typically the ideal mass required; for significantly different fragment sizes, scale the mass accordingly [e.g., ˜6-18 ng for a 1,500-bp PCR product]). For further details, see How much DNA to use in a sequencing reaction? and Estimate template input by agarose gels.
  • Sample volume:  Provide 1-3 µl of PCR product per tube (or well); the actual volume submitted needs to be the amount required for one sequencing reaction (see above). All samples should have the same total volume of liquid; if needed, equalize sample volumes with either commericial nuclease-free water or 10 mM Tris.
  • High Concentration PCR products:  If even 1 µl of the original PCR product would exceed the desired template input, dilute an aliquot of the PCR product with either commericial nuclease-free water or 10 mM Tris so that you can provide 1-3 µl of appropriately diluted PCR product per tube (or well).
  • Multiple Primer reactions?:  If you want to sequence the PCR products with more than one primer, you must provide one set of templates for each primer. For example, to have your templates sequenced in both directions, you need to provide a duplicate set of each template so that the forward primer can be used with one set and the reverse primer with the other set.
  • Label Submission:  Upon successful submission of your online request, the 'Submission #" will appear in your 'Submission History'. Then, label the tube rack itself with:
    • Submission #;
    • Date;
    • PI's Last Name; and,
    • Submitter name or initials.
  • Delivery site:  Transfer tubes to a Genomics Core rack (taking your own rack back to your lab), and put samples in the Mini-fridge (by sink) in the Genomics Core (A628 Life Science Annex).

    [Note: If the Core is locked, put your samples in the 'After-Hours' Mini-fridge located by the ice machine in LSA 624 (Common Room).]

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