Monthly Archives: December 2016

DNA extraction – naturally bleached Porites spp.

I was able to collect some naturally bleached Porites astreoides and Porites porites specimens while in Belize in November. I hope to use these as reference samples without symbiont DNA for RADseq. These samples were flash frozen instead of preserving in SS-DMSO. To extract DNA, a small fragment was crushed with a mortar and pestle and divided between three 1.5 ml tubes for extraction using the Qiagen DNeasy assay.  An overnight proteinase K 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 100-200 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, 125 volts for 30 min.

sample ng/ul (qubit) total vol total DNA ng
Past_bleached_1 15.8 100 1580
Past_bleached_2 7.02 100 702
Past_bleached_3 6.32 100 632
Ppor_bleached_1 16.7 100 1670
Ppor_bleached_2 6.74 200 1348
Ppor_bleached_3 13.7 100 1370


DNA looks great but note that RNase was not used and there may be some RNA in there. Also, note that the pellet was brown and there may be other contaminants.