help with DNA extraction

[bev at tissugen.com.au]

Joyce C. Faler wrote:

> What kind of tissue do you extract?  Are you using fresh tissue or preserved? 
> We extract fish tissue preserved in ethanol or lysis buffer.  Sometimes we'll
> get a group of tissue samples (fin clips from fish) that are faded out (the
> normal pigment looks washed out), and our extracted product will be an oily
> brown droplet that will not amplify.  We have gotton to hate seeing faded
> looking fin clip tissue because
> no matter what we do, we can't get it to amplify, even though the extract
> product looks great on an agarose gel.  We have varied the extraction process
> with no success:  chloroform/isoamyl alcohol; saturated NaCl; Quiagen kit; and
> Wizard Cleanup kit on extracted products.

> Anyway, in our case, we suspect something in the initial tissue preservation
> process that occurs in outdoor field circumstances.

> We look forward to anyone who can help you (and perhaps us) out.
> -Joyce Faler
> Fish Genetics Lab
> University of Idaho, Aquaculture Research Institute

> bev at tissugen.com.au wrote:

Hi Joyce,
We are extracting human tissue (prostate and kidney) which is taken at time of operation and frozen at -80 degrees until extraction. However, your oily brown droplet which looks OK on agarose but will not amplify - that sounds exactly the same as the extraction product I am now getting!

You mention that your tissues are sometimes preserved in lysis buffer, and during tissue digestion our samples can spend up to 48hrs in lysis buffer (10mM Tris pH8; 100mM EDTA and 0.5% SDS, with added Proteinase K) at 55 degrees. However, I have no idea yet what the problem is but I'll let you know if I find out!

Bev Shannon
Tissugen Pty Ltd,
Perth, Western Australia. 


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