DNA extraction: Anthopleura elegantissima

In the interest of comparing methylation levels between symbiotic states in Anthopleura elegantissima, I extracted DNA from three zooxanthellate and three zoochlorellate individuals. These were anemones that were collected last summer at Point Lawrence, Orcas Island, and had been residing in indoor sea tables at Shannon Point since then. For each specimen, I excised part of the tentacle crown with scissors and deposited the tissue directly into a microfuge tube. I opted to freeze the tissue in the -80ºC freezer since earlier attempts with fresh tissue did not seem as effective (i.e., the tissue seemed resistant to lysis). After a day or two in the freezer, I pulled the samples out, rinsed them with PBS, and proceeded with the Qiagen DNeasy assay.  After addition of proteinase K, I used a small pestle to homogenize the sample. An overnight lysis period at 56ºC was used. DNA was eluted via two passes with 50 µl AE buffer (100 µl total volume). To further clean the DNA, samples were subject to ethanol precipitation using this protocol. Samples were re-eluted in 50 µl AE buffer.

To assess DNA quantity and quality, samples were tested with the Qubit BR DNA assay followed by electrophoresis on a 1% agarose gel with 1X TBE, 135 volts for 25 min.

sample ng/µl ng/µl post-dilution (100 µl buffer) total DNA
Ae-B-1 256 128 12800
Ae-B-2 183 91.5 9150
Ae-B-3 304 152 15200
Ae-G-1 163 81.5 8150
Ae-G-2 166 83 8300
Ae-G-3 274 137 13700


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