PCR inhibition from worm DNA preparation

Deanne Bell dbell at fresno.ars.usda.gov
Thu Jun 17 13:03:09 EST 2004


Hi Craig

I work with plants & insects and often run into PCR inhibition:
I utilize several DNA protocols all of which have been modified for
microtube extractions:
CTAB; QIAGEN DNeasy Plant kit;

Wang X.D., Wang Z.P., and Zou Y.P., 1996. An improved isolation of
nuclear DNA from leaves of wild grapevine dried with silica gel. Plant
Mol. Biol. Reporter 14: 369-373.

My latest favorite for problematic tissue is a modification from:
Tabaeizadeh Z., Agharbaoui Z., Harrak H., and Poysa V., 1999. Transgenic
tomato plants expressing a Lycopersicon chilense chitinase gene
demonstrate improved resistance to Verticillium dahliae race 2. Plant
Cell Reports 19: 197-202.

Briefly:  Tissue is homogenized in microtube using a bead mill with
extraction buffer (sodium bisulfate omitted). Nuclei centrifuged -
supernatant discarded. Pellet resuspended in Lytic Buffer. Incubated at
65*C. Lysate extracted twice with chloroform:octanol (24:1). DNA
precipitated with isopropanol. Pellet washed with ethanol then dissolved
in TE. 

Hope this helps
TTFN
Dee Bell


> -----Original Message-----
> From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]
> On Behalf Of Craig Wilding
> Sent: Thursday, June 17, 2004 8:23 AM
> To: methods at hgmp.mrc.ac.uk
> Subject: PCR inhibition from worm DNA preparation
> 
> I have extracted genomic DNA from earthworms and compost worms but am
> struggling to PCR from this. It seems to contain a PCR inhibitor as
> adjudged by spiking PCRs that definitely work with this DNA. I have
> cleaned it up with a Promega wizard column which has not completely
> cured the problem. I don't know what the inhibitor is but have read
that
> DNA extracted from soil can contain PCR inhibiting humic and fulvic
> acids. Since the worms had soil in their guts this may be the source
of
> the problem. Has anyone any ideas for how to clean the DNA and
> completely get rid of the inhibitors?
> Thanks,
> Craig.
> 
> --
> Craig Wilding
> Genetics Department
> Westlakes Research Institute
> International Research and Graduate Centre
> Westlakes Science & Technology Park
> Moor Row
> Cumbria
> CA24 3JY
> 
> Tel 	01946 514158
> Fax 	01946 514166
> e-mail 	craig.wilding at westlakes.ac.uk
> 
> 
> 
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