[Annelida] Working with Formalized Tissue...Help!!

Elena Kupriyanova via annelida%40net.bio.net (by lena.kupriyanova from gmail.com)
Mon Mar 2 19:03:19 EST 2009


Dear Emily,

It is not easy to say the least, so DNeasy kit clearly would not help.

See, for example 
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VGB-4D3W313-3&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=81f2530ea398b346e41433df5a24323b

Boyle, E.E. Zardus J.D.,^ Chase M.R. Etter R.J. and Rex M.A.^  2004. 
Strategies for molecular genetic studies of preserved deep-sea 
macrofauna. Deep-sea Res. Part 1. Ocean Res Pap 51: 1319-1336.

Abstract

With the development of new methods to sequence DNA from preserved 
organisms, existing archival collections can be used to document the 
population genetic structure of deep-sea species. This has made possible 
the first direct inferences about patterns of evolutionary 
diversification in the soft-sediment macrofauna. Here we report 
protocols and success rates for amplifying and sequencing regions of the 
mitochondrial 16S rDNA, Cytochrome oxidase I (COI), and Cytochrome b 
(cytb) genes from formalin-fixed protobranch bivalves and gastropods, 
major components of the deep-sea benthos. DNA was extracted from 1532 
individuals of 12 common bathyal and abyssal species that had been fixed 
in formalin and preserved in alcohol for up to 36 years. DNA was also 
extracted from 53 individuals that were dried upon collection, some of 
which were collected more than 100 years ago. The overall success rate 
for amplification by PCR was 44%, but this varied considerably among 
species, stations, and cruises. When DNA amplified, sequencing success 
was generally high, averaging 85% and ranging from 19% to 100%. The 
reliability of amplification and sequencing depend strongly on how 
samples are treated during collection and storage. Amplification success 
was similar among samples collected from the same station and samples 
collected on the same cruise. We provide recommendations on strategies 
for primer design, PCR, and sample selection to improve success rates 
for genetic analysis of preserved deep-sea organisms. The success rates 
from different collections, sampling stations, and cruises provide 
important guidance for selecting material for future genetic work on 
deep-sea collections examined here.


wormlab wrote:
> Hi All,
>
> I’m looking for help getting DNA out of formalized worms. I’ve been 
> trying to do DNA extractions using the Qiagen DNeasy Kit and I’m not 
> getting anything. I’ve tried bathing the samples in two 15 minute 
> washes of room temperature PBS before extraction, but that hasn’t 
> worked either. If anyone has worked with formalized worms and has any 
> advice, your help is greatly appreciated.
>
> Thanks!
>
> Emily Krause
> Zoology Department
> University of Hawaii
> Honolulu, HI 96822
> (808) 956-5794
>
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