help with DNA extraction

[Joyce C. Faler]

Bev,
We preserve our tissues for months or years at a time in lysis buffer without the proteinase K, then add proteinase K to finish tissue breakdown at 55°C.  We also have tissues that are frozen or in alcohol that we remove to a lysis solution containing proteinase K for digestion from 3 - 16 hours (ie overnight).  I
look forward to any solutions you find!
-Joyce

bev at tissugen.com.au wrote:

> 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.
>
> http://biowww.net/mynews/tree.php?group_name=bionet.molbio.methds-reagnts&begin=0