Genomic DNA from Sea Cucumber

Bill Burnett w.j.burnett at ncl.ac.uk
Tue Nov 3 03:28:53 EST 1998



leerc wrote:

> Hi,
>
> We are having problem of extracting Genomic DNA from Sea Cucumber
> (H.edulis, S.Varigatus and others). Anyone who have sucessfully extracted
> these genomic DNA would be very much appreciate if can provide us the
> protocol.
>
> Thank you for any information.
>
> C.S.NG
> Bio Diagnostics Sdn Bhd
> Malaysia
> e-mail: "C.S.NG"<leerc at pc.jaring.my>

Echinoderms are all b******s.  Sure you wouldn't rather work on fish? I hear
fish are wonderful.  Trouble is fish people think everything works as well as
fish...  Am I bitter?
Of course not....

You could try my brittle star protocol (Applied Environmental Microbiology
63: 1721-1724) which works with most material, or Williams and Benzie
(Molecular Ecology 6:559-573).

Echinoderms seem to be the DNAase champions of the world - you have to knock
them out immediately or all you'll ever see is a nasty degraded smear.
Preservation in ethanol or DMSO within seconds of death works.  If you're
using frozen material then NEVER let it thaw - grind it in liquid N2 and tip
the frozen powder directly into a tube with PCI and buffer.  Don't try and
proteinase frozen tissue, you'll get nothing...

There may be some nice echinoderms out there that are no problem but I've
never met one...

You're next problem having gotten decent DNA is to get it to amplify.
E'derms are also PCR inhibition world champions.  Most of them will work fine
after a number of washes through a 100kDal MW filter (but do this BEFORE you
precipitate the DNA).  Some of them just never worked at all for me...

Good luck...

Bill (now working on cnidarians - piece of cake by comparison...)
-----------------------------------------------------------------------
Dr. W. Burnett, University of Newcastle.   w.j.burnett at ncl.ac.uk
Throw the XXXX away to reply - drink real beer in stead.





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