Does anybody know if the chemical composition of marine and freshwater fish muscle tissue have differences that can influence on DNA properties when muscle tissue is preserved in ethanol?
Can't get PCR-ready DNA for COI gene from muscles of freshwater fishes with alkaline lysis.
Thank you.
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I suppose it should be more clear, sorry. There is no doubt that ethanol is a great preservation agent. But when I try to implement ethanol-preserved freshwater fish muscle tissue in HotSHOT DNA isolation method I can not get good results in opposite to marine fish muscles.
The question is related to different reaction to preservation, I suppose. But I can not get usefull "keyword" for an answer)
I've got good results with other DNA isolation methods but what is wrong with HotSHOT when implementing on freshwater fishes with ethanol-based preservation?
I have been using ethanol preservation for marine fish muscle tissue with great success and know many others in associated labs that say the same. I haven't noticed any differences in ethanol vs. DMSO preservation for my fish tissues. Generally speaking researchers used ethanol for fish muscle tissue for decades, and I wouldn't expect there to be significant changes to chemical composition that would affect PCR.
The African Center for DNA Barcoding (ACDB) has been collecting both marine and freshwater fish muscle tissue for the last 5 years and have gotten very good results assessing the CO1 gene.
Is it possible you are extracting some polymerase inhibitors along with your sample? We have had this problem with crustacean samples but not really with fish. Have you tried diluting the primers and re-running. It does sound a bit weird and I also cannot see why it would not work with freshwater samples. We have also preserved tissue in high-salt DMSO solution as an alternative to ethanol but this was to deal with issues of shipping flammable ethanol. However, rather than trying different presertvatives I would investigate thoroughly your extraction and amplification method first.
Also are you using a kit? Sometimes you can get different results making up your own solutions although the kits should be pretty good by now - at least this allows you to check your problems are not due to the kit.
We obtained good results when fish tissues were preserved in absolute alcohol. Then the tissues were homogenized in TES buffer followed by DNA extraction using Phenol-Chloroform- Isoamylalcohol method.
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