DNA extraction from fish.

snowdon at waikato.ac.nz snowdon at waikato.ac.nz
Tue Jul 14 19:13:25 EST 1992


In article <Pine.2.4.9207092110.A7859 at milton.u.washington.edu>, walterh at U.WASHINGTON.EDU (Walter Hill) writes:
> Dear fellow lab rats:
> We've been trying to get DNA out of Fish (most Salmon and mostly muscle
> but some liver, too) and have had seemingly miserable success.  We've
> tried almost everything from the old proteinase-phenol-chloroform routine
> to commercial kits such as A.S.A.P. and the like.  Tried disrupting tissue
> with freeze-thawing and homogenizing, etc.
> Anybody care to contribute a method that is performing well in their hands?
> Any (well, almost any) suggestions welcome.
> I'll summarize responses and post it to the net.
> Many thanks in advance.
> Regards, -Walt Hill
> 
> walterh at u.washington.edu (primary)           (secondary) WEH at FDACFSAN.BITNET
> Food & Drug Admin., Seafood Products Research Center, Bothell, WA 98041-3012
> VOICE:(206) 486-8788           "Cook it first"            (206) 483-4996:FAX 


I have routinely extracted DNA from rainbow trout liver & muscle using a
modification of a protocol for DNA extractions from plants (J.J. & J.L. 
Doyle, 1990: Isolation of plant DNA from fresh tissue.  FOCUS 12; pp13-
15).  This is a hot-CTAB method which I've found has given excellent yield
(particaularly from liver), is relatively simple and works almost every 
time!  The technique goes like this:

About 200-500mg tissue (fresh or -70 deg.C frozen) is ground gently
in 5ml of CTAB isolation buffer (100mM Tris-HCl, 10mM EDTA, 1.4M NaCl, 
2% (w/v) CTAB (hexadecyltrimethyl-ammonium bromide), and 0.2% (v/v) 
2-mercaptoethanol) pre-heated to 60 deg.C, in a 15ml Wheaton glass 
grinding tube.  (Alternatively, tissue can be ground in liquid Nitrogen 
and added directly to heated CTAB buffer in a 10ml Eppendorf - this 
works just as well).

Transfer to 10ml Eppendorf, mix gently and incubate at 60 deg.C for 30min.

Extract once with an equal volume of chloroform:isoamyl alcohol (24:1) and
spin for 5 mins (4000rpm).

Remove aqueous phase to new 10ml Eppendorf tube using a wide-bore 
pippette and precipitate nucleic acids by adding 2/3 volumes of cold 
(-20 deg.C) isopropanol (or you can use 99% EtOH) and swirling gently.
You generally get large quantities of nucleic acids visible at this point.

Spin 5-10 mins at 4000rpm and decant of supernatant (the pellet is 
relatively hard, so supernatant be poured off quite easily).

Wash pellet at least 30mins (can be left overnight) by gentle shaking
in 10ml wash buffer (76% EtOH; 10mM ammonium acetate) - I just sit my
tubes on a tilting or rotating shaker plate.

Supernatant is poured off and the pellet air-dried for about 30 mins 
(drops of liquid can be removed from the inside of the tube with a 
pippette), and the pellet is resuspended in 0.2-0.5ml TE (10mM Tris-HCl
(pH7.4), 1mM EDTA).  The pellet usually takes a day or so to dissolve
thoroughly.

Electrophoresis at this stage generally reveals the presence of RNA and
mitDNA as well as nuclear genomic DNA, but I've had no problems with
restriction digestion at this point.  You can continue with proteinase 
and/or RNAase treatment but I've never needed to bother.


I can't see any reason why this method shouldn't be transferable from 
rainbow trout to other salmonids, but I have also used another 
method with no problems that was passed on to me by John Taggart from
Queen's University of Belfast, Nthn Ireland.  He has just recently
(late 1991 or early this year - don't know the issue off-hand) 
published it as a note in the Journal of Fish Biology.  It's also a 
pretty easy procedure - you can do loads of samples in a single day - 
and it works for small quantities of liver, muscle or adipose fin in
most salmonid species.  (The big advantage I had with it was that I 
was able to get adipose fin samples from live fish, also fins can be 
stored in 95% EtOH so you don't have to worry about freezing - quite 
useful if collecting out in the wops!).

John's method is basically a small-scale proteinase/phenol/chl'form
procedure, so may be similar to what you've tried already, but it 
may be worth a read anyway.


Good luck!!!


Rod Snowdon


snowdon at waikato.ac.nz
Biological Sciences, University of Waikato, Hamilton, New Zealand



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