Yesterday and today I made a salt saturated DMSO solution for coral DNA preservation in the field. I found support for this solution as a preservative for corals based on Gaither et al. 2011 and Concepcion et al. 2014. There is also an older article by Seutin et al. 1991 that appears to have initially promoted this solution for animal DNA preservation.

On the web I found this protocol for making salt saturated DMSO, and this is what I followed.

First I made 1L 0.5M EDTA:

900 ml nanopure H20
EDTA (FW 292.24) 146.12 g
NaOH pellets to pH EDTA to 8.0
Brought final volume to 1L

I ended up adding LOTS of NaOH pellets to bring the pH up since I did not have disodium EDTA. This took a few hours.

Then for the salt saturated DMSO:

0.5M disodium EDTA 1L
Dimethyl Sulfoxide (DMSO) 400mL
Nanopure water 500mL
NaCl – Enough to saturate the solution.

First I added 300 g NaCl, then another 100 g , then another 20 g. I brought the volume up to 2 L with nanopure water. Some solid NaCl remained, which was left out when I transferred the solution to a container. I then autoclaved the solution.

Continuing the extraction from yesterday, samples were centrifuged at 4,000 x g for 1.5 min to pellet DNA. The ethanol was aspirated and the samples were air dried under the hood for about an hour (they were watched to ensure they were not over dried). DNA was resuspended in 0.2 ml nanopure water.

Here is the electrophoresis protocol I used:

1. 75 ml TAE with 0.65 g agarose was microwaved for 1 min (with cap resting on 100 ml pyrex jar, watching for boil over) to heat and facilitate dissolution of the agarose. The jar was swirled with cap on. Solution was re-heated for a few more seconds to further facilitate particulate dissolution.

2. Solution left to cool without lid for 1-2 min

3. 7.5 ul EtBr added and swirled to mix

4. Solution left to cool for another minute before slowly pouring into the gel tray. The gel tray included an 8 slot comb, and the gel solution was poured in the opposite end from the comb.

5. Gel was left for 30 min to cool and harden.

6. Comb was removed and gel tray was rotated 90 degrees to align with electrodes.

7. Gel chamber filled with enough 1x TAE to just cover gel (~1 mm depth over gel).

8. Gel loaded with 5 ul green ladder (GeneRuler 100 bp) on each end. 20 ul of each sample was mixed with 3 ul purple loading dye on a sheet of parafilm and loaded into gel.

9. Electrodes plugged in and power supply set to 120 V.

10. Gel was run for 55 min, when ladder reference dye was ~1cm from edge of gel.

There were no bands present for either sample. Perhaps the DNA was degraded; the samples were collected 5 months ago. I did notice that both DNA pellets were quite brown in color.

Today I extracted DNA from the Porites astreoides samples I collected back in May. The samples consisted of a single coral colony that I relocated from the lagoon to the backreef, and I sampled the coral on the day of transplantion (t = 0) and three days later (t = 3). The samples were kept under refrigeration in ethanol. I extracted the DNA using DNAzol.

The DNAzol protocol was as follows:

1. The surface layer of each coral core was scraped with a razor blade and the tissue/skeletal material was homogenized with a sterile plastic pestle in a microfuge tube with 0.5 ml DNAzol.

2. The homogenate was sedimented by centrifugation at 10,000 x g for 10 min. The supernatant was then transferred to a new tube.

3. DNA was precipitated by the addition of 0.25 ml 100% ethanol. Samples were mixed by inversion and I was able to spool the DNA and transfer to a new tube.

4. DNA was washed twice with 1 ml 75% ethanol. A quick spin was used each time to pellet the DNA before removing the supernatant.

5. Samples were stored under refrigeration in 95% ethanol.

Well, it’s the start of another quarter here, so what better time to jot down some goals for the next month? I head to Belize in November for some field work, so my primary October goal is to prepare for this trip. This includes having a solid list of tasks to make the best of my time there, which I hope to have my committee review for any feedback they might have. This month I would also like to take a stab at RADseq, and possibly EpiRADseq, two methods I hope to employ to characterize the population genetics and epigenetics of Porites astreoides in Belize.