Services – EtOH-EDTA Precipitation

Standard DNA Purification (maximum recovery)

When the goal is to recover the maximum amount of DNA, including the tiniest fragments, the Genomics Core uses the protocols listed at How to clean DNA templates for sequencing? To use this option, select "Std. DNA EtOH Precip. (max-recovery): 1-96 samples" from the "Services" request page,.

These protocols are highly effective and inexpensive for situations in which recovery of small products is at least not a problem. Further, in many cases, these protocols will even adequately deplete PCR samples of residual primers and primer-dimers.

However, some primers are so resistant to removal that the opposite primer (vs. the primer added to the sequencing reaction) will remain in high enough concentrations to generate either excessive noise or even signal intensities equivalent to those coming from the 'sequencing' primer.

Thus, for direct sequencing of PCR products, clients should usually request one of the 'PCR Primer depletion' protocols below.

PCR Primer depletion

While many PCR primers are amenable to removal by the 'standard' method, others are not. Thus, the Genomics Core has optimized an EtOH-EDTA precipitation protocol specifically for depleting the residual original primers and primer-dimers from PCR products in preparation for Sanger-Sequencing reactions.

The protocol is based upon sub-optimizing the normal EtOH-EDTA precipitation protocol sufficiently enough to avoid recovering small fragments (e.g., <100-bp). Thus, it comes in two 'flavors':
– PCR Primer depletion (<69% A/T templates): EtOH Precip.; and,
– PCR Primer depletion (>70% A/T templates): EtOH Precip.

Two 'flavors' are required because templates with extremely high A/T content are already difficult to precipitate and a strong sub-optimization may lose the desired fragments. By contrast, for templates where G/C content exceeds ~30%, small products might be precipitated unless the protocol is sub-optimized to a strong degree.

These protocols have been implemented successfully with numerous submissions, retaining fragments ≥100 bp and effectively removing residual primers and primer-dimers. Nevertheless, if you have never had a GC 'primer-depletion' EtOH-EDTA protocol used before on your templates, please consider submitting a small test-run before requesting processing of full plates.

Online Submission Request

Make the necessary 'selections'.
Quantity: '1' covers up to 96-samples (based on idea of submitting up to 96 samples in a single PCR plate) – although small numbers of samples (e.g., perhaps up to ~32) can be submitted in strip tubes.
In the "Client Memo" box, specify the desired resupension volume (if any) and any other relevant information. [ Note : If the DNA is resuspended , the Core normally uses TVLE (10 mM Tris, 0.05 mM EDTA).]
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 must be submitted in either 8-strip tubes (0.2-ml) or 96-well plates.

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).]