This morning I was able to use the Nanodrop to quantitate the DNA in the A. elegantissima heat stress samples. The results corroborate the earlier results on the gel, showing low concentrations overall but slightly higher concentrations in samples from Exp 2. Sample #4 from Experiment 2 had the highest DNA concentrations, but the concentrations were still only 4-5 ng/ul.

All samples are in 230 ul water.

I extracted DNA from the Anthopleura elegantissima heat stress experiment samples using DNAzol. This was done on 2/24/15 for samples from experiment 1, and on 3/3/15 for samples from experiment 2. See previous posts for details of these experiments.

The DNAzol protocol was as follows:

1. Tentacles were homogenized with a sterile plastic pestle in a microfuge tube with 0.5 ml DNAzol. I tried to apply as few strokes as possible, but anemone tentacles are very tough and are difficult to homogenize even after being frozen.

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. A cloudy precipitate was generally observed, but few samples had noticeable threads of DNA formed. Thus, spooling was not attempted.

4. Samples were centrifuged at 4,000 x g for 1.5 min to pellet DNA. Supernatant was then aspirated.

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

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

7. On 4/3/15, 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).

8. DNA was resuspended in 0.2 ml nanopure water.

Yesterday (4/3/15), I finally got around to running a gel to take a look at the DNA. Many thanks to Jake for showing me where things were and getting me started. Here is the electrophoresis protocol I followed:

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 a 12 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 20 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.

Sample abbreviations: L = ladder, A = ambient, H = heated, 5 = 5°C, 37 = 37°C

The very faint bands suggest very low concentrations of DNA; experiment 2 samples had darker bands, but they were still faint. Experiment 2 was performed in part because I suspected that not enough DNA was obtained from the 1-2 tentacles collected in experiment 1. So experiment 2 should have higher concentrations of DNA, as seen here, but apparently even more tissue would have been better. There is 0.18 ml sample remaining.

I decided to redo the Anthopleura elegantissima thermal stress experiment for two reasons. First, I was not totally happy with the lab conditions leading up to the first experiment. The animals did not have good water flow or regular water changes, the temperature was a bit too low, and the light conditions were artificial. The animals also both died following the stress. Secondly, upon extracting the DNA from those samples, the yield seemed low simply based on visual assessment.

So, yesterday (3/1/15) I redid the experiment at Shannon Point, this time taking two anemones directly from the sea tables and placing them in a 35C water bath for one hour. I decided not to have the water bath quite as warm as in the earlier experiment since it seemed a bit excessive (and it killed the animals). I also clipped a few more tentacles than before so there would be more tissue mass for DNA extraction. Tentacles were clipped before and after the thermal stress and immediately placed in a freezer at -80C. As in the previous experiment, the thermal stress caused the anemones to contract and release their pedal disk from the glass substrate. This morning, however, the anemones had reattached and were even slightly expanded in the flow-through seawater system. It seems likely they will survive the stress.

Today I performed a thermal stress experiment on Anthopleura elegantissima, with the goal of comparing the methylome and transcriptome before and after the stress.

Two specimens of A. elegantissima were brought to the lab from the sea tables at Shannon Point Marine Center. These individuals were originally collected from Lawrence Point, Orcas Island, WA, in late June 2014. They were brought to the Roberts Lab on 2/3/2015, and were kept in a small plastic container with about 1L of seawater, which was changed weekly. The container was held inside a well-lit refrigerator kept at 5C.

Today, after the specimens had spent two weeks under these conditions, I clipped a few tentacles from each of them before transferring them into beakers filled with seawater, which were then placed inside a water bath at 37C for one hour. After this thermal stress, I again clipped a few tentacles. Before and after tentacle samples were immediately placed in the -80C freezer. The samples were placed in a freezer box on the bottom shelf of the freezer.

The larger of the two anemones was labelled as “#1″, while the smaller one was labelled “#2″. After the heat shock, both anemones had detached from the watch glasses they had been attached to, indicating that they were clearly affected by the shock. The larger of the two anemones appeared slightly healthier after the shock; I suspect it is the one more likely to survive if there is any mortality.

2/23/2015 Update

I came in to find that both anemones had died. They were not heavily decomposed so either they died recently, or were kept in good shape by the cold water temperature.

2/24/2015 Updata

I perfomed DNAzol isolation of DNA on the samples. Only two of the samples showed an obvious precipitate, but because of the generally low quantities I opted to pellet the DNA instead of spooling it. I left the samples in 95% ethanol and plan to quantitate the DNA next week.

Today I finished validating a CpG O/E workflow Steven put together by analyzing the C. gigas transcriptome and comparing it to the results of Gavery and Roberts (2010). The results agreed well. Notably, there was a similar bimodal distribution of CpG O/E, and major gene categories showed similar levels of CpG O/E (e.g., cell adhesion around 0.7 and DNA metabolism around 0.5).

Since mid-Oct 2014 I have been getting familiar with blasting transcriptomes using IPython. Steven helped me get set up with the first blast of Madracis and I’ve taken it from there. IPython notebooks can be found below.

Madracis auretenra

Pocillopora damicornis

Porites astreoides