We have lobster larvae (possibly slipper lobsters and some spiny lobsters) that were collected over a period of 2 years and stored in 96-99% ethanol, and have tried to extract DNA from them (trying pereiopod tissue or thorax punch) using both the zymo insect DNA and the zymo universal g-DNA extraction kits.
Initially, we were able to obtain DNA and amplify COI, however, the sequences returned were very messy and terminated abruptly. Thereafter, we have failed to get COI to amplify. Does anyone know of a good method for larval DNA extraction, to obtain good quality DNA for downstream applications?
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042757 Check this out. :)
Another way can be to fix the pleopods again in 100 % ethanol twice for 5 min, twice in xylol for 5 min and let them dry for 20 min at room temperature. After that grind it with e.g. TissueLyser or a similar equippment and use the ATL buffer + proteinase K (Qiagen) over nigt shaking them at 56°C (according to the manual). Then you will get a nice DNA useful for PCR. :-)
Hi Johan,
I doubt that your problem is the quality of DNA. After extraction it should not degrade that quickly, or did you store the extracted DNA at room temperature? I rather suggest that you have some problems in the PCR. Did you BLAST the short fragments? Maybe all you amplified and sequenced was some kind of contamination.
I successfully extracted DNA with the hotshot method (see paper) using muscular tissue. The method is super quick, easy and cheap. Also I suggest to try other COI primers or ITS as this usually amplifies very well (see the second paper for some other primers). I often had problem with standart COI primers in Crustacea.
Best,
Martin
Another problem can be the ethanol itself. I also had this when we stored fish organs in it. After 48 - 72 hours the PCR was successful but from the same materials after 4 weeks it got negative by PCR. The DNA concentration was the same. Clear, I went for viral DNA which also can be that the irregular distribution of the virus in the organ can be guilty. Therefore we used one trunk kidney from carp in 20 pieces a 30 µg for extraction. The other one we stored in ethanol (100%). The second kidney was negative after 4 weeks in ethanol. Due to a recommendation of a colleague we changed to isopropanol which solved the storage problem. Now we still have positive signals by PCR after a storage of 5 year and longer. I know in crustaceans it is generally to store the parts in ethanol but think about that as well.
Best wishes
Sven
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