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Subject: DNA in Fucus
From: Jennifer Chaseley <[log in to unmask]>
Reply-To:[log in to unmask]
Date:Thu, 24 May 2001 08:36:39 GMT0BST
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Dear All

I have had several requests to post the protocol which I found to be
successful on the web. It is quite long, takes about 2 days but its
definately worth it, the genomic DNA I extracted was suitable for
PCR applications and so Obviously got rid of alot of the chemicals
within seaweed which have been reported to kill the Taq.

It was supplied by several people but particular thanks to Jim, for
his step by step guide, and several others who provided the
theoretical background for this approach.

The method is summarised below, please forgive some of the
unscientific terms.

Best of Luck
Jenny

Saweed which had been washed in sterile seawater and then
frozen at -70oC was ground to a fine powder under liquid nitrogen.

To each gram of material ( I used 2g and got good results) 10mls of
2x CTAB buffer was added. (100mM Tris pH 8.0, 1.5M NaCl, 50mM
EDTA and 2% CTAB and 0.1% PVPP) The buffer and  seaweed
were mixed and SDS added to a final concentration of 0.1% and
0.2% mercaptoethanol, this was left at room temperature for 2
hours.

An equal volume of chloroform: Isoamylalcohol ( 24:1) was added
and spun at 2000g for 10 minutes. The aqueos phase was removed
and washed in CI as before, this was repeated until the interface
was clean. The aqueous phase was precipitated with 2/3 volume of
cold isopropanol for 2-3 hours at -20oC mixing gently ( not
vortexing). This was spun at 3K for 25 minutes. The pellet was
washed with 70% ethanol and allowed to air dry.

2mls of TE was added and left in the fridge O/N.

1g of CsCl was added per ml of TE and spun at 25K for 25 minutes
to removed any precipitating polysaccharides. The supernatant was
removed to a fresh tube and a drop of triton-X and ethidium Bromide
to a final concentration of 100ug/ml was added. the sample was
spun in an ultracentrifuge for 9.5 hours at 60 000RPM

Tube was viewed under UV light and the ethidium bromide
containing ring was removed ( top ring is protein, pellet is DNA and
debris middle ring is DNA) and washed in water saturated butanol
until all
the EtBr had been removed. 2 vols of TE containing 0.2M NaCl was
addedand
mixed with a further 2.5 vols of EtOH and allowed to precipitate
overnight
at -20oC. The DNA wqas pelleted by spinning for 30minutes at top
speed in
a microfuge. The pellet was washed in 70% EtOH and allowed to
air dry.
Pellet was resuspended in 50uL of TE at 65oC and then analysed
on a gel!!!

Good luck,

I also got sent a paper with an alternative method which I haven't
tried: DNA Isolation Protocol for Red Seaweed ( Wattier RA et al,
Plant Molecular Biology Reporter 18:275-281, 2000) which utilises
proteinase K and not CTAB.

Jenny Chaseley
School of Biosciences
Cardiff University

029 20 876800
07803 552376

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