[Taxacom] Storage of specimens in ethanol

Richard Clopton rclopton at oakmail.peru.edu
Thu Nov 13 13:34:11 CST 2008


Donat Agosti asked about using adulterated ethanol for storage of specimens for later DNA extraction.
 
Truth is that it depends on two things: how much DNA the specimen contains and how long a sequence you want to eventually recover.
 
Adulterated alcohols tend to chew up DNA. On extraction, you end up with small fragments rather than high molecular weight DNA. In my experience, things like MEK chew your samples down to about 300-700 bp fragments. If you are after long regions (ssu, lsu) or single copy genes, this is problemmatic if the sample is small. In my experience, some portion of the DNA survives without too much damage - if you have a lot of tissue to work with, you'll still be able to pull long PCR/ single copy genes but it will be much more difficult because you have a lot of fragment scraps to contend with (there are ways, but why go there if you don't have to do so?). But if you don't have much tissue to work with, probability is against being able to pull anything longer then 300-500 bp. (I'm a protistologist - 1 protist = 1 genomic copy so I don't have much room for error.) This is still okay if you are going after short sections, i.e. just ITS1 or ITS2: they're multicopy and smaller than your fragmentation size.
 
There is another alternative worth considering. The zoology side of the house tends towards ethanol storage. But the truth is you're just trying to make the sample anhydrous in an effort to shut down DNAase activity and prevent microbial colonization. The botany side of the house has traditionally gotten good results using dried material.  When I started working out of country and good ethanol became problemmatic (air travel), I started just drying my specimens at 55-60 C in the sample tube I would later use for digestion to start the extraction. There are lots of ways to generate clean heat in the field: light bulbs, heated sand in a coffee cup, glass beads in a beaker on a small hot plate, etc. It works like a charm and I get better DNA (higher quantity AND less fragmentation). If I read your web site correctly Donat, you're working ants. Try drying your samples down in a microcentrifuge tube: break the exoskeleton open to allow moisture to escape, pop them in an open top tube, heat at 55-60 C. I suspect they'll dry down nicely in no time at all. You have so many cells to work with that just removing legs to a sample tube and drying them down would probably give you enough tissue to get a good DNA sample and they'd dry much faster. Try it with something local or in colony as a test, but I suspect it will work fine. If you can't get them to dry down fast enough, carry a small bottle of human consumption high-proof spirits (I've used both vodka and everclear), dehydrate the specimen in spirits first, then blot and heat dry. Sounds odd, but DNA drys well without structural damage and when dry is an incredibly stable molecule.
 
Regards, 
 
Rich Clopton
Peru State College
Peru, Nebraska USA
rclopton at oakmail.peru.edu 
 
Dr. Richard E. Clopton, PhD
Associate Editor, Comparative Parasitology
Professor of Biology
Downey Family Honors Chair for Science
 

Post:
Dr. R. E. Clopton
Department of Natural Science
101 Hoyt Hall, 600 Hoyt Street
Peru State College
Peru, Nebraska  68421  USA
 
TEL: 402/872-2284
FAX: 402/872-2375
EM: rclopton at oakmail.peru.edu 
WWW: http://science.peru.edu/gregarina




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