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. [However, one might still be using the same amount of PCR product because only ~50% of the treated sample is used in a sequencing reaction.]
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, specify ...
- 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
Tubes: Samples exceeding ~25 ul should be submitted in 0.5-1.5 ml tubes (standard or Eppendorf LoBind); otherwise, 0.2-ml tubes are acceptable.
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 plastic "After-Hours" GC drop-box located in the adjacent Cold Room (beside sink, Rm. A650).]
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