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Subject: Re: DNA extraction from fish eggs
From: stefano mariani <[log in to unmask]>
Reply-To:Scientific forum on fish and fisheries <[log in to unmask]>
Date:Mon, 14 Nov 2011 12:43:21 +0000
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Dear all,
given the very numerous requests for feedback, I have now collated below a list of answers that I got.
In respect of privacy, I have maintained anonymity.

Thanks again.

Stf.

>>>>>>>>
Hi

The problem sounds similar we had with frog eggs. Two possible solutions if the fish eggs work like frogs eggs.

1. Suck the egg contents out with a pipette inserted trough mucus. This is the method we used successfully with common frogs to isolate DNA for our work

2. Get rid of the mucus with 2.6% L-cysteine. I do not know if this works with fish mucus, but it effectively dejellied our frog eggs as described in the enclosed paper.

I hope this helps,
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Hi Stefano,

Have you tried extraction methods that will digest away a lot of the PCR inhibitors?  I've had good luck extracting from snails using a standard CTAB protocol (with a Phenol-Chloroform-Isoamyl alchohol wash). 

I'm also attaching a paper that is specifically for small tissues/ embryonic fish.. which might work.

Good luck!
___________________________________

Dear Stefano,
how are you?  Have you tried CTAB extraction?  That usually works quite well with "slimy stuff".
____________________________________

Stephano,

I wondered if it would be useful to check with protocols for extracting DNA from cacti and other mucilaginous plants.  Seems that there may be protocols that get rid of various proteins and carbohydrates in plants that are transferable.
__________________________________

I think you would try PROGEMA WIZARD DNA (salt-precipitation method) and/or DNA advance (magnetic beads method):
 We use them at our lab on different kinds of tissues (we use the chelex INSTAGENE too, not recommended for tissues with too much mucus)

http://www.promega.com/resources/protocols/technical-manuals/0/wizard-genomic-dna-purification-kit-protocol/
http://www.beckmangenomics.com/products/dna_purification_and_cleanup/agencourt_dnadvance.html

Im attaching a few papers regarding your question...
Best and good luck,
__________________________________

For invertebrates, especially slimy ones, you can use any derivation of the CTAB methods. This is good when you have polysaccharides and you want to get ride of them.

cheers
__________________________________

Hello Stefano,

In the past, while trying to extract DNA from slugs (Deroceras reticulatum), that are covered in mucus, we faced a similar problem as you describe. We successfully extracted DNA by using the standard technique of digesting the tissue with proteinase K, and then using as many chloroform/phenol steps as necessary to remove all protein and other stuff from the solution. Usually two chloroform/phenol steps were enough, and invariably the method gave good quality DNA. Try to remove as much mucus as possible from the eggs (freezing them may make that easier?), and the chloroform/phenol steps will take care of the rest.
____________________________________

Hi Stefano,

You could try simple detergent. I've heard that can help in snail slima etc. (never tried myself, though) Good luck!

BAHL A & PFENNINGER M (1996) A rapid method for isolation of DNA using laundry detergent. Nucleic Acids Research 24, 1587-1588.
_____________________________________

Hello Stephano
I used cyanure potassium to wash the jelly of amphibian eggs. Very useful, all the jelly is distroyed and you obtain a clear isolated embryo. Perhaps similar effect with this kind of fish eggs
best
_____________________________________

Hi Stefano,

Probably wont be of much use but I know common carp (among other species) farmers regularly use tannin/tannic acid to 'wash' carp eggs when using the hypophysation technique to artificially spawn offspring. It removes the adhesive properties (i.e. mucus) of the eggs thus making them easier to handle, incubate etc. Prolonged exposure apparently damages the egg but a quick wash would appear to be ok. May be worth a look, sorry I cant be of more help.
______________________________________

hi Stefano,
Prior to DNA extraction, you can add NaCl to the tissue to a final concentration of 0.7M, and 10% w/v of CTAB solution, and incubated at 70 degrees C for 10 minutes.
I hope this helps.
_____________________________________

I used to work with Stegastes partitus eggs, but never had trouble with the chemicals in the mucus. I was doing microsatellites. The eggs of this species are really tiny. Do you find dirty DNA?

I used the Wizard DNA extraction kit. The individual eggs were digested whole in 100 L of Nuclei Lysis Solution and 2 l of Proteinase K (20 mg/mL), and the Wizard kit protocol was scaled down by a factor of five.

I also extracted eggs using the silica-based 96-well plate extraction protocol, from Elphinstone et al. 2003. I also reduced the concentrations by a factor of five.

Cheers,
________________________________

Stefano
The relative stickiness of eggs is often related to pH (lower the pH, the more sticky the eggs), so an alkaline solution may allow you to remove the mucus?

Hope all is good.
________________________________

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